Use of complement pathway inhibitors to treat ocular diseases

ABSTRACT

The present invention relates to the treatment of ocular diseases and conditions by administering a complement pathway inhibitor, particularly an alternative pathway inhibitor. Ocular diseases include age-related macular degeneration, diabetic M retinopathy, and ocular angiogenesis. One embodiment comprises the administration of an anti-Factor D antibody in the form of a whole antibody, a Fab fragment or a single domain antibody. Other complement component inhibitors that may be useful in the present method include Factor H or inhibitors that block the action of properdin, factor B, factor Ba, factor Bb, C2, C2a, C3a, C5, C5a, C5b, C6, C7, C8, C9, or C5b-9.

This application is a continuation of U.S. application Ser. No.14/270,848, filed May 6, 2014, now abandoned, which is a continuation ofU.S. application Ser. No. 12/092,346, filed Oct. 9, 2008, now U.S. Pat.No. 8,753,625, which is a National Stage application of InternationalApplication No. PCT/US2006/043103, filed Nov. 4, 2006, which claims thebenefit of U.S. Provisional Application No. 60/733,763 filed Nov. 4,2005, the disclosure of each of which is incorporated by referenceherein in their entireties.

FIELD OF THE INVENTION

This invention relates to the inhibition of the complement pathway,particularly Factor D, in patients suffering from ocular relatedconditions and diseases associated with complement activation such asage-related macular degeneration, diabetic retinopathy.

BACKGROUND OF THE INVENTION

Macular degeneration is a clinical term that is used to describe afamily of diseases that are characterized by a progressive loss ofcentral vision associated with abnormalities of the Bruch's membrane,the choroid, the neural retina and/or the retinal pigment epithelium. Inthe center of the retina is the macula lutea, which is about ⅓ to ½ cm.in diameter. The macula provides detailed vision, particularly in thecenter (the fovea), because the cones are higher in density. Bloodvessels, ganglion cells, inner nuclear layer and cells, and theplexiform layers are all displaced to one side (rather than restingabove the ones), thereby allowing light a more direct path to the cones.Under the retina is the choroid, a collection of blood vessels embeddedwithin a fibrous tissue, and the pigmented epithelium (PE), whichoverlays the choroid layer. The choroidal blood vessels providenutrition to the retina (particularly its visual cells). The choroid andPE are found at the posterior of the eye.

The retinal pigment epithelial (RPE) cells, which make up the PE,produce, store and transport a variety of factors that are responsiblefor the normal function and survival of photoreceptors. Thesemultifunctional cells transport metabolites to the photoreceptors fromtheir blood supply, the chorio capillaris of the eye. RPE cells alsofunction as macrophages, phagocytizing the tips of the outer segments ofrods and cones, which are produced in the normal course of cellphysiology. Various ions, proteins and water move between the RPE cellsand the interphotoreceptor space, and these molecules ultimately effectthe metabolism and viability of the photoreceptors.

Age-related macular degeneration (AMD), the most prevalent maculardegeneration, is associated with progressive loss of visual acuity inthe central portion of the visual field, changes in color vision, andabnormal dark adaptation and sensitivity. Two principal clinicalmanifestations of AMD have been described as the dry, or atrophic, form,and the wet, or exudative, form. The dry form is associated withatrophic cell death of the central retina or macula, which is requiredfor fine vision used for activities such as reading, driving orrecognizing faces. About 10-20% of these dry AMD patients progress tothe second form of AMD, known as wet AMD.

Wet (neovascular/exudative) AMD is caused by abnormal growth of bloodvessels behind the retina under the macula and vascular leakage,resulting in displacement of the retina. hemorrhage and scar formation.This results in a deterioration of sight over a period of months toyears. however, patients can suffer a rapid loss of vision. All wet AMDcases are originated from advanced dry AMD. The wet form accounts for85% of blindness due to AMD. In wet AMD, as the blood vessels leak fluidand blood, scar tissue is formed that destroys the central retina.

The most significant risk factors for the development of both forms areage and the deposition of drusen, abnormal extracellular deposits,behind the retinal pigment epithelium. Drusen causes a lateralstretching of the RPE monolayer and physical displacement of the RPEfrom its immediate vascular supply, the choriocapillaris. Thisdisplacement creates a physical barrier that may impede normalmetabolite and waste diffusion between the choriocapillaris and theretina. Drusen are the hallmark deposits associated with AMD. Thebiogenesis of drusen involves RPE dysfunction, impaired digestion ofphotoreceptor outer segments, and subsequent debris accumulation. Drusencontain complement activators, inhibitors, activation-specificcomplement fragments, and terminal pathway components, including themembrane attack complex (MAC or C5b-9), which suggests that focalconcentration of these materials may produce a powerful chemotacticstimulus for leukocytes acting via a complement cascade (Killingsworth,et al., (2001) Exp Eye Res 73, 887-96). recent studies have implicatedlocal inflammation and activation of the complement cascade in theirformation (Bok D. Proc Natl Acad Sci (USA). 2005; 102: 7053-4; Hademan GS, et al. Prog Retin Eye Res. 2001; 73: 887-96).

Wet AMD is associated with choroidal neovascularization (CNV) and is acomplex biological process. Pathogenesis of new chloroidal vesselformation is poorly understood, but such factors as inflammation,ischemia, and local production of angiogenic factors are thought to beimportant. Although inflammation has been suggested as a playing a role,the role of complement has not been explored. A preliminary study of CNVhas been shown to be caused by complement activation in a mouse model(Bora P S, J Immunol. 2005; 174: 491-497).

The complement system is a crucial component of the innate immunityagainst microbial infection and comprises a group of proteins that arenormally present in the serum in an inactive state. These proteins areorganized in three activation pathways: the classical, the lectin, andthe alternative pathways (V. M. Holers, In Clinical Immunology:Principles and Practice, ed. R. R. Rich, Mosby Press; 1996, 362-391).Molecules on the surface of microbes can activate these pathwaysresulting in the formation of protease complexes known asC3-convertases. The classical pathway is a calcium magnesium-dependentcascade, which is normally activated by the formation ofantigen-antibody complexes. It can also be activated in anantibody-independent manner by the binding of C-reactive proteincomplexed with ligand and by many pathogens including gram-negativebacteria. The alternative pathway is a magnesium-dependent cascade whichis activated by deposition and activation of C3 on certain susceptiblesurfaces (e.g. cell wall polysaccharides of yeast and bacteria, andcertain biopolymer materials).

The alternative pathway participates in the amplification of theactivity of both the classical pathway and lectin pathway (Suankratay,C., ibid; Farries, T. C. et al., Mol. Immunol. 27: 1155-1161(1990).Activation of the complement pathway generates biologically activefragments of complement proteins, e.g. C3a, C4a and C5a anaphylatoxinsand C5b-9 membrane attack complexes (MAC), which mediate inflammatoryresponses through involvement of leukocyte chemotaxis, activation ofmacrophages, neutrophils, platelets, mast cells and endothelial cells,increased vascular permeability, cytolysis, and tissue injury.

Factor D may be a suitable target for the inhibition of thisamplification of the complement pathways because its plasmaconcentration in humans is very low (1.8 μg/ml), and it has been shownto be the limiting enzyme for activation of the alternative complementpathway (P. H. Lesavre and H. J. Müller-Eberhard. J. Exp. Med., 1978;148: 1498-1510; J. E. Volanakis et al., New Eng. J. Med., 1985; 312:395-401). The inhibition of complement activation has been demonstratedto be effective in treating several disease indications using animalmodels and in ex vivo studies, e.g. systemic lupus erythematosus andglomerulonephritis (Y. Wang et al., Proc. Natl. Acad. Sci.; 1996, 93:8563-8568).

Using single-nucleotide polymorphism (SNP) analysis of AMD patients, aFactor H genetic variant (Y402H) was found to be highly associated withincreased incidence of AMD (Zareparsi S, Branham KEH, Li M, et al. Am JHum Genet. 2005; 77: 149-53; Hains J L, et al. Sci 2005; 208: 419-21).Persons who are either homozygous or heterozygous for this pointmutation of Factor H gene may account for 50% of AMD cases. Factor H isthe key soluble inhibitor of the alternative complement pathway(Rodriguez de Cordoba S, et al. Mol Immunol 2004; 41: 335-67). It bindsto C3b and thus accelerates the decay of the alternative pathwayC3-convertase (C3bBb) and acts as a co-factor for the Factor I-mediatedproteolytic inactivation of C3b. Histochemical staining studies showthat there is similar distribution of Factor H and MAC at theRPE-choroid interface. Significant amounts of deposited MAC at thisinterface found in AMD patients indicate that the Factor H haplotype(Y402H) may have attenuated complement inhibitory function. It isspeculated that Factor H (Y402H) may have a lower binding affinity forC3b. Therefore, it is not as effective as wild type Factor H ininhibiting the activation of the alternative complement pathway. Thisputs RPE and choroids cells at sustained risk for alternativepathway-medicated complement attack.

It had been shown that lack of Factor H in plasma causes uncontrolledactivation of the alternative pathway with consumption of C3 and oftenother terminal complement components such as C5. In keeping with thisfinding, plasma levels of Factor H are known to decrease with smoking, aknown risk Factor for AMD (Esparza-Gordillo J, et al., Immunogenetics.2004; 56: 77-82).

Currently, there is no proven medical therapy for dry AMD, and notreatments available for advanced dry AMD. In selected cases of wet AMD,a technique known as laser photocoagulation may be effective for sealingleaky or bleeding blood vessels. Unfortunately, laser photocoagulationusually does not restore lost vision, but merely slows, and in somecases, prevents further loss. Recently, photodynamic therapy has shownto be effective in stopping abnormal blood vessel growth in about onethird of wet AMD patients when treated early. In Visdyne PhotodynamicTherapy (PDT), a dye is injected into the patient's eye, it accumulatesin the area of vessel leakage in the retina and, when exposed to a lowpower laser, it reacts sealing off the leaking vessels. In addition tothese two laser techniques, there are several anti-angiogenesistherapies targeting vascular endothelial growth Factor (VEGF) beingdeveloped for the treatment of wet AMD. However, only 10% treatedpatients show vision improvement.

In view of these inadequate treatments for wet AMD and the total lack oftreatments available for advanced dry AMD, there is a clear need for thedevelopment of new treatments for this serious disease. Our inventionprovides a novel approach to treating this serious disease.

SUMMARY OF THE INVENTION

The present invention relates to complement inhibitors for the treatmentof ocular related conditions or diseases, such as age-related maculardegeneration (AMD), diabetic retinopathy, ocular angiogenesis (such asocular neovascularization affecting choroidal, corneal, or retinaltissue), and the ocular conditions involving complement activation.treatment of AMD includes both the dry and we forms of AMD.

The complement inhibitors of the present invention include, but are notlimited to, those inhibiting the alternative complement pathway, such asFactor D, properdin, Factor B, Factor Ba, and Factor Bb, and theclassical complement pathway, such as C3a, C5, C5a, C5b, C6, C7, C8, C9and C5b-9. The present invention also includes the use of complementinhibitors in combination with other agents, such as anti-angiogenicagents and anti-inflammatory agents such as steroids.

Another embodiment of the present invention relates to the use of C5aRand C3aR inhibitors, such as antibodies and derived fragments and signaldomain constructs, as well as small molecule compounds.

Another embodiment of the present invention relates to the use ofrecombinant soluble CR1 (TP10) and its derived proteins; use of C3inhibiting molecules (such as Compstatin, a peptidomimetic and binds andinhibits C3 activation); siRNAs that block the synthesis of C3, C5, FD,factor P, factor B

these inhibitors can be, but not limited to, small molecule chemicalcompounds, nucleotides, peptides, proteins, peptidomimetics andantibodies.

Another embodiment of the present invention includes the use of humanFactor H purified from human blood or recombinant human Factor Hadministered to patients intraocularly or by any other clinicallyeffective route.

Antibodies of the present invention include whole immunoglobulins, scFv,Fab, Fab′, Fv, F(ab′)2, or dAb. Domain antibodies comprise either a VHdomain or a VL domain.

One embodiment of the present invention is the use of a monoclonalantibody which binds to Factor D and blocks its ability to activate thealternative complement pathway. Such antibodies are described in WO01/70818 and US 20020081293, which are incorporated here by reference,such as monoclonal antibody 166-32 produced from the hybridoma depositedwith the American Type Culture Collection (ATCC), 12301 Parklawn Drive,Rockville, Md. 20852, U.S., on Feb. 24, 1998, and designated HB12476.The present invention also includes antibodies that specifically bind tothe same epitope as monoclonal antibody 166-32. Monoclonal antibodies ofthe present invention may also include the humanized antibodies ofco-pending application No. 60/856,505 filed Nov. 2, 2006, which isincorporated herein by reference.

One embodiment of the present invention is the use of a monoclonalantibody which binds to complement component C5a. Such antibodiesinclude antibody 137-26 produced from the hybridoma deposited with theATCC and designated PTA-3650, and any antibody that specifically bindsto the same epitope as 137-26.

According to the present invention, the complement pathway inhibitor maybe administered by (a) parenteral administration; (b) biocompatible orbioerodable sustained release implant; (c) implantation of an infusionpump; or (d) local administration, such as subconjunctivaladministration or by intravitreal administration. The complementinhibitor may also be administered by parenteral administration selectedfrom oral administration, enteral administration and topicaladministration. Topical administration may include an eye wash solution,an eye ointment, an eye shield or an eye drop solution.

In addition the complement inhibitor of the present invention may beadministered in combination with a immunomodulatory or immunosuppressivecompound.

Another embodiment of the present invention relates to theadministration of nucleic acid constructs that are capable of expressingthe complement pathway inhibitors for gene therapy.

Another embodiment of the present invention includes a method forscreening for complement inhibitors that are useful in the treatment ofAMD comprising the use of an AMD model in senescent Ccl-2 orCcr-2-deficient mice. These mice manifest similar histopathologicalchanges found in human dry and wet AMDs. These mice may be treated withcomplement inhibitors or Factor H intravitreally. Histologicalexamination may be performed to determine protection from AMDdevelopment in mice treated with the agents to be tested.

DETAILED DESCRIPTION OF THE INVENTION

This invention is not limited to the particular methodology, protocols,cell lines, vectors, or reagents described herein because they may vary.Further, the terminology used herein is for the purpose of describingparticular embodiments only and is not intended to limit the scope ofthe present invention. As used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural reference unless thecontext clearly dictates otherwise, e.g., reference to “a host cell”includes a plurality of such host cells.

Unless defined otherwise, all technical and scientific terms and anyacronyms used herein have the same meanings as commonly understood byone of ordinary skill in the art in the field of the invention. Althoughany methods and materials similar or equivalent to those describedherein can be used in the practice of the present invention, theexemplary methods, devices, and materials are described herein.

All patents and publications mentioned herein are incorporated herein byreference to the extend allowed by law for the purpose of describing anddisclosing the proteins, enzymes, vectors, host cells, and methodologiesreported therein that might be used with the present invention. However,nothing herein is to be construed as an admission that the invention isnot entitled to antedate such disclosure by virtue of prior invention.

Definitions

The term “amino acid sequence variant” refers to polypeptides havingamino acid sequences that differ to some extent from a native sequencepolypeptide. Ordinarily, amino acid sequence variants will possess atleast about 70% homology, or at least about 80%, or at least about 90%homology to the native polypeptide. The amino acid sequence variantspossess substitutions, deletions, and/or insertions at certain positionswithin the amino acid sequence of the native amino acid sequence.

The term “identity” or “homology” is defined as the percentage of aminoacid residues in the candidate sequence that are identical with theresidue of a corresponding sequence to which it is compared, afteraligning the sequences and introducing gaps, if necessary to achieve themaximum percent identity for the entire sequence, and not consideringany conservative substitutions as part of the sequence identity. NeitherN- or C-terminal extensions nor insertions shall be construed asreducing identity or homology. Methods and computer programs for thealignment are well known in the art. Sequence identity can be readilycalculated by known methods, including but not limited to thosedescribed in (Computational Molecular Biology, Lesk, A. M., ed., OxfordUniversity Press, New York, 1988; Biocomputing: Informatics and Genomeprojects, Smith, D. W., ed., Academic Press, New York, 1993; Computer,Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G.,eds., Humana Press, New Jersey, 1994; Sequence Analysis in MolecularBiology, von Heinje, G., Academic Press, 1987; and Sequence AnalysisPrimer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York,1991; and Carillo, 30 H., and Lipman, D., SIAM J. Applied Math., 48:1073 (1988). Methods to determine identity are designed to give thelargest match between the sequences tested. Computer program methods todetermine identity between two sequences include, but are not limitedto, the GCG program package (Devereux, J., et al., Nucleic AcidsResearch 12(1): 387 (1984)), BLASTP, BLASTN, and FASTA (Atschul, S. F.et al., J Molec. Biol. 215: 403-410 (1990). The BLAST X program ispublicly available from NCBI and other sources (BLASTManual, Altschul,S., et al, NCBI NLM NIH Bethesda, Md. 20894, Atschul, S., et al., J.Mol. Biol. 215: 403-410 (1990). The wall-known Smith Waterman algorithmmay also be used to determine identity.

The term “antibody” herein is used in the broadest sense andspecifically covers intact monoclonal antibodies, polyclonal antibodies,multispecific antibodies (e.g., bispecific antibodies) formed from atleast two intact antibodies, and antibody fragments, so long as theyexhibit the desired biological activity.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. In contrast to polyclonal antibody preparations whichinclude different antibodies directed against different determinants(epitopes), each monoclonal antibody is directed against a singledeterminant on the antigen. The modifier “monoclonal” indicates thecharacter of the antibody as being obtained from a substantiallyhomogeneous population of antibodies, and is not to be construed asrequiring production of the antibody by any particular method. Forexample, the monoclonal antibodies to be used in accordance with thepresent invention may be made by the hybridoma method first described byKohler et al, Nature, 256:495 (1975), or may be made by recombinant DNAmethods (see, e.g., U.S. Pat. No. 4,816,567). The “monoclonalantibodies” may also be isolated from phage antibody libraries using thetechniques described in Clackson et al., Nature, 352:624-628 (1991) andMarks et al., J. Mol. Biol., 222:581-597 (1991), for example.

The monoclonal antibodies herein specifically include “chimeric”antibodies in which a portion of the heavy and/or light chain isidentical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired biological activity (U.S. Pat. No. 4,816,567; and Morrison etal, Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984).

“Antibody fragments” comprise a portion of an intact antibody comprisingthe antigen-binding or variable region thereof. Examples of antibodyfragments include Fab, Fab′, F(ab′)₂, and Fv fragments; diabodies;linear antibodies; single-chain antibody molecules; and multispecificantibodies formed from antibody fragment(s).

An “intact” antibody is one which comprises an antigen-binding variableregion as well as a light chain constant domain (C_(L)) and heavy chainconstant domains, C_(H)1, C_(H)2 and C_(H)3. The constant domains may benative sequence constant domains (e.g. human native sequence constantdomains) or amino acid sequence variant thereof. The intact antibody mayhave one or more effector functions.

Antibody “effector functions” refer to those biological activitiesattributable to the Fc region (a native sequence Fc region or amino acidsequence variant Fc region) of an antibody. Examples of antibodyeffector functions include C1q binding; complement dependentcytotoxicity; Fc receptor binding; antibody-dependent cell-mediatedcytotoxicity (ADCC); phagocytosis; down regulation of cell surfacereceptors (e.g. B cell receptor; BCR), etc.

Depending on the amino acid sequence of the constant domain of theirheavy chains, intact antibodies can be assigned to different “classes”.There are five-major classes of intact antibodies: IgA, IgD, IgE, IgG,and IgM, and several of these may be further divided into “subclasses”(isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy-chainconstant domains that correspond to the different classes of antibodiesare called α, δ, ϵ, γ, and μ, respectively. The subunit structures andthree-dimensional configurations of different classes of immunoglobulinsare well known.

“Antibody-dependent cell-mediated cytotoxicity” (ADCC) refers to acell-mediated reaction in which nonspecific cytotoxic cells that expressFc receptors (FcRs) (e.g. Natural Killer (NK) cells, neutrophils, andmacrophages) recognize bound antibody on a target cell and subsequentlycause lysis of the target cell. The primary cells for mediating ADCC, NKcells, express FcγRIII only, whereas monocytes express FcγRI, FcγRII andFcγRIII. FcR expression on hematopoietic cells. To access ADCC activityof a molecule of interest, an in vitro ADCC assay, such as thatdescribed in U.S. Pat. No. 5,500,362 or 5,821,337 may be performed.Useful effector cells for such assays include peripheral bloodmononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively,or additionally, ADCC activity of the molecule of interest may beassessed in vivo, e.g., in a animal model. Several such models areavailable.

The term “variable” refers to the fact that certain portions of thevariable domains differ extensively in sequence among antibodies and areused in the binding and specificity of each particular antibody for itsparticular antigen. However, the variability is not evenly distributedthroughout the variable domains of antibodies. It is concentrated inthree segments called hypervariable regions both in the light chain andthe heavy chain variable domains. These hypervariable regions are alsocalled complementarity determining regions or CDRs. The more highlyconserved portions of variable domains are called the framework regions(FRs). The variable domains of native heavy and light chains eachcomprise four FRs, largely adopting a β-sheet configuration, connectedby three hypervariable regions, which form loops connecting, and in somecases forming part of, the β-sheet structure. The hypervariable regionsin each chain are held together in close proximity by the FRs and, withthe hypervariable regions from the other chain, contribute to theformation of the antigen-binding site of antibodies (see Kabat et al.,Sequences of Proteins of Immunological Interest, 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md. (1991)).

The term “hypervariable region” when used herein refers to the aminoacid residues of an antibody which are responsible for antigen-binding.the hypervariable region generally comprises amino acid residues from a“complementarity determining region” or “CDR” (e.g., residues 24-34(L1), 50-56 (L2) and 89-97 (L30 in the light chain variable domain and31-35 (H1), 50-65 (H2) and 95-102 (H3) in the heavy chain variabledomain; Kabat et al., Sequences of Proteins of Immunological Interest,5th Ed. Public Health Service, National Institutes of Health, Bethesda,Md. (1991)) and/or those residues from a “hypervariable loop” (e.g.,residues 2632 (L1), 50-52 (L2) and 91-96 (L3) in the light chainvariable domain and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavychain variable domain; Chothia and Lesk J. Mol. Biol. 196:901-917(1987). “Framework Region” or “FR” residues are those variable domainresidues other than the hypervariable region residues as herein defined.

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fc” fragment, whose name reflects its ability tocrystallize readily. Pepsin treatment yields an F(ab′)₂ fragment thathas two antigen-binding sites and is still capable of cross-linkingantigen.

The Fab fragment also contains the constant domain of the light chainand the first constant domain (CH1) of the heavy chain. Fab′ fragmentsdiffer from Fab fragments by the addition of a few residues at thecarboxy terminus of the heavy chain CH1 domain including one or morecysteines from the antibody hinge region. Fab+-SH is the designationherein for Fab′ in which the cysteine residue(s) of the constant domainsbear at least one free thiol group. F(ab′)₂ antibody fragmentsoriginally were produced as pairs of Fab′ fragments which have hingecysteins between them. Other chemical couplings of antibody fragmentsare also known.

The “light chains” of antibodies from any vertebrate species can beassigned to one of two clearly distinct types, called kappa (κ) andlambda (λ), based on the amino acid sequences of their constant domains.

“Fv” is the minimum antibody fragment which contains a completeantigen-recognition and antigen-binding site. This region consists of adimer of one heavy chain and one light chain variable domain in tight,non-covalent association. It is in this configuration that the threehypervariable regions of each variable domain interact to define anantigen-binding site on the surface of the V_(H). V_(L) dimer.Collectively, the six hypervariable regions confer antigen-bindingspecificity to the antibody. However, even a single variable domain (orhalf of an Fv comprising only three hypervariable regions specific foran antigen) has the ability to recognize and bind antigen, although at alower affinity than the entire binding site.

“Single-chain Fv” or “scFv” antibody fragments comprise the V_(H) andV_(L) domains of antibody, wherein these domains are present in a singlepolypeptide chain. Preferably, the Fv polypeptide further comprises apolypeptide linker between the V_(H) and V_(L) domains which enables thescFv to form the desired structure for antigen binding. For a review ofscFv see Plückthun in The Pharmacology of Monoclonal Antibodies, vol.113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315(1994). Anti-ErbB2 antibody scFv fragments are described in WO93/16185;U.S. Pat. No. 5,571,894; and U.S. Pat. No. 5,587,458.

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which comprise a variable heavy domain (V_(H))connected to a variable light domain (V_(l)) in the same polypeptidechain (V_(H)V_(L)). By using a linker that is too short to allow pairingbetween the two domains on the same chain, the domains are forced topair with the complementary domains of another chain and create twoantigen-binding sites. Diabodies are described more fully in, forexample, EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl.Acad. Sci. USA, 90:6444-6448 (1993).

A “single-domain antibody” is synonymous with “dAb” and refers to animmunoglobulin variable region polypeptide wherein antigen binding iseffected by a single variable region domain. A “single-domain antibody”as used herein, includes i) an antibody comprise heavy chain variabledomain (VH), or antigen binding fragment thereof, which forms an antigenbinding site independently of any other variable domain, ii) an antibodycomprising a light chain variable domain (VL), or antigen bindingfragment thereof, which forms an antigen binding site independently ofany other variable domain, iii) an antibody comprising a VH domainpolypeptide linked to another VH or a VL domain polypeptide (e.g., VH-VHor VHx-VL), wherein each V domain forms an antigen binding siteindependently of any other variable domain, and iv) an antibodycomprising VL domain polypeptide linked to another VL domain polypeptide(VL-VL), wherein each V domain forms an antigen binding siteindependently or any other variable domain. As used herein, the VLdomain refers to both the kappa and lambda forms of the light chains.

“Humanized” forms of non-human (e.g., rodent) antibodies are chimericantibodies that contain minimal sequence derived from non-humanimmunoglobulin. Humanized antibodies are human immunoglobulins whereinthe hypervariable regions are replaced by residues from a hypervariableregion of a non-human species, such as mouse, rat, rabbit or nonhumanprimate having the desired specificity, affinity, and capacity. In someinstances, framework region (FR) residues of the human immunoglobulinare replaced by corresponding non-human residues. Furthermore, humanizedantibodies may comprise residues that are not found in the humanantibody or in the non-human antibody. These modifications are made tofurther refine antibody performance. In general, the humanized antibodywill comprise substantially all of at least one, and typically two,variable domains, in which all or substantially all of the hypervariableloops correspond to those of a non-human immunoglobulin and all orsubstantially all of the FRs are those of a human immunoglobulinsequence. The humanized antibody optionally also will comprise at leasta portion of an immunoglobulin constant region. (Fc), typically that ofa human immunoglobulin. Examples of humanization technology may be foundin, e.g., Queen et al. U.S. Pat. Nos. 5,585,089, 5,693,761; 5,693,762;and 6,180,370, which are incorporated herein by reference.

Antibody Generation

The antibodies of the present invention may be generated by any suitablemethod known in the art. The antibodies of the present invention maycomprise polyclonal antibodies. Methods of preparing polyclonalantibodies are known to the skilled artisan (Harlow, et al., Antibodies:a Laboratory Manual, (Cold spring Harbor Laboratory Press, 2nd ed.(1988), which is hereby incorporated herein by reference in itsentirety).

For example, antibodies may be generated by administering an immunogencomprising the antigen of interest to various host animals including,but not limited to, rabbits, mice, rats, etc., to induce the productionof sera containing polyclonal antibodies specific for the antigen. Theadministration of the immunogen may entail one or more injections of animmunizing agent and, if desired, an adjuvant. Various adjuvants may beused to increase the immunological response, depending on the hostspecies, and include but are not limited to, Freund's (complete andincomplete), mineral gels such as aluminum hydroxide, surface activesubstances such as lysolecithin, pluronic polyols, polyanions, peptides,oil emulsions, keyhole limpet hemocyanins, dinitrophenol, andpotentially useful human adjuvants such as BCG (bacille Calmette-Guerin)and Corynebacterium parvum. Additional examples of adjuvants which maybe employed include the MPL-TDM adjuvant (monophosphoryl lipid A,synthetic trehalose dicorynomycolate). Immunization protocols are wellknown in the art in the art and may be performed by any method thatelicits an immune response in the animal host chosen. Adjuvants are alsowell known in the art.

Typically, the immunogen (with or without adjuvant) is injected into themammal by multiple subcutaneous or intraperitoneal injections, orintramuscularly or through IV. The immunogen may include an antigenicpolypeptide, a fusion protein or variants thereof. Depending upon thenature of the polypeptides (i.e., percent hydrophobicity, percenthydrophilicity, stability, net charge, isoelectric point etc.), it maybe useful to conjugate the immunogen to a protein known to beimmunogenic in the mammal being immunized. Such conjugation includeseither chemical conjugation by derivatizing active chemical functiongroups to both the immunogen and the immunogenic protein to beconjugated such that a covalent bond is formed, or throughfusion-protein based methodology, or other methods known to the skilledartisan. Examples of such immunogenic proteins include, but are notlimited to, keyhole limpet hemocyanin, ovalbumin, serum albumin, bovinethyroglobulin, soybean trypsin inhibitor, and promiscuous T helperpeptides. Various adjuvants may be used to increase the immunologicalresponse as described above.

The antibodies useful in the present invention comprise monoclonalantibodies. Monoclonal antibodies may be prepared using hybridomatechnology, such as those described by Kohler and Milstein, Nature,256:495 (1975) and U.S. Pat. No. 4,37,110, by Harlow, et al.,Antibodies: A Laboratory Manual, (Cold spring Harbor Laboratory Press,2.sup.nd ed. (1988), by Hammerling, et al., Monoclonal Antibodies andT-Cell Hybridomas (Elsevier, N.Y., (1981)), or other methods known tothe artisan. Other examples of methods which may be employed forproducing monoclonal antibodies include, but are not limited to, thehuman B-cell hybridoma technique (Kosbor et al., 1983, Immunology today4:72; Cole et al., 1983, Proc. Natl. Acad. Sci. USA 80:2026-2030), andthe EBV-hybridoma technique (Cole et al., 1985, Monoclonal AntibodiesAnd Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Such antibodies maybe of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and anysubclass thereof. The hybridoma producing the antibodies of thisinvention may be cultivated in vitro or in vivo.

Using typical hybridoma techniques, a host such as a mouse, a humanizedmouse, a mouse with a human immune system, hamster, rabbit, camel or anyother appropriate host animal, is typically immunized with an immunogento elicit lymphocytes that produce or are capable of producingantibodies that will specifically bind to the antigen of interest.Alternatively, lymphocytes may be immunized in vitro with the antigen.

Generally, in making antibody-producing hybridomas, either peripheralblood lymphocytes (“PBLs”) are used if cells of human origin aredesired, or spleen cells or lymph node cells are used if non-humanmammalian sources are desired. The lymphocytes are then fused with animmortalized call line using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell (Goding, MonoclonalAntibodies: Principles and Practice, Academic Press, (1986), pp.59-103). Immortalized call lines are usually transformed mammaliancells, particularly myeloma cells of rodent, bovine or human origin.Typically, a rat or mouse myeloma cell line is employed. The hybridomacells may be cultured in a suitable culture medium that preferablycontains one or more substances that inhibit the growth or survival ofthe unfused, immortalized cells. For example, if the parental cells lackthe enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT orHPRT), the culture medium for the hybridomas typically will includehypoxanthine, aminopterin, and thymidine (“HAT medium”), substances thatprevent the growth of HGPRT-deficient cells.

Preferred immortalized cell lines are those that fuse efficiently,support stable high level expression of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. More preferred immortalized cell lines are murine myeloma lines,which can be obtained, for instance, from the Salk Institute CellDistribution Center, San Diego, Calif. and the American Type CultureCollection, Manassas, Va. human myeloma and mouse-human heteromyclomacell lines may also be used for the production of human monoclonalantibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur el al.,Monoclonal Antibody Production Techniques and Applications, MarcelDekker, Inc., New York, (1987) pp. 51-63).

The culture medium in which the hybridoma cells are cultured can then beassayed for the presence of monoclonal antibodies directed against theimmunogen. The binding specificity of monoclonal antibodies produced bythe hybridoma cells is determined by, e.g., immunoprecipitation or by anin vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linkedimmunoadsorbant assay (ELISA). Such techniques are known in the art andwithin the skill of the artisan. The binding affinity of the monoclonalantibody can, for example, be determined by a Scatchard analysis (Munsonet al., Anal. biochem., 107:220 (1980)).

After the desired hybridoma cells are identified, the clones may besubcloned by limiting dilution procedures and grown by standard methods(Goding, supra). Suitable culture media for this purpose include, forexample, Dulbecco's Modified Eagle's medium and RPMI-1640. Themonoclonal antibodies secreted by the subclones may be isolated orpurified from the culture medium by conventional immunoglobulinpurification procedures such as, e.g., protein A-sepharose,hydroxyapatite chromatography, gel exclusion chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

A variety of methods exist in the art for the production of monoclonalantibodies and thus, the invention is not limited to their soleproduction in hydridomas. For example, the monoclonal antibodies may bemade by recombinant DNA methods, such as those described in U.S. Pat.No. 4,816,567. In this context, the tem “monoclonal antibody” refers toan antibody derived from a single eukaryotic, phage, or prokaryoticclone. The DNA encoding the monoclonal antibodies of the invention canbe readily isolated and sequenced using conventional procedures (e.g.,by using oligonucleotide probes that are capable of binding specificallyto genes encoding the heavy and light chains of murine antibodies, orsuch chains from human, humanized, or other sources). The hydridomacells of the invention serve as a preferred source of such DNA. Onceisolated, the DNA may be placed into expression vectors, which are thentransformed into host cells such as NS0 cells, Simian COS cells, Chinesehamster ovary (CHO) cells, or myeloma cells that do not otherwiseproduce immunoglobulin protein, to obtain the synthesis of monoclonalantibodies in the recombinant host cells. The DNA also may be modified,for example, by substituting the coding sequence for human heavy andlight chain constant domains in place of the homologous murine sequences(U.S. Pat. No. 4,816,567; Morrison et al, supra) or by covalentlyjoining to the immunoglobulin coding sequence all or part of the codingsequence for a non-immunoglobulin polypeptide. Such a non-immunoglobulinpolypeptide can be substituted for the constant domains of an antibodyof the invention, or can be substituted for the variable domains of oneantigen-combining site of an antibody of the invention to create achimeric bivalent antibody.

The antibodies may be monovalent antibodies. Methods for preparingmonovalent antibodies are well known in the art. For example, one methodinvolves recombinant expression of immunoglobulin light chain andmodified heavy chain. The heavy chain is truncated generally at anypoint in the Fc region so as to prevent heavy chain cross-linking.Alternatively, the relevant cysteine residues are substituted withanother amino acid residue or are deleted so as to preventcross-linking.

Antibody fragments which recognize specific epitopes may be generated byknown techniques. For example, Fab and F(ab′)2 fragments of theinvention may be produced by proteolytic cleavage of immunoglobulinmolecules, using enzymes such as papain (to produce Fab fragments) orpepsin (to produce F(ab′)2 fragments). F(ab′)2 fragments contain thevariable region, the light chain constant region and the CH1 domain ofthe heavy chain.

For some uses, including in vivo use of antibodies in humans and invitro detection assays, it may be preferable to use chimeric, humanized,or human antibodies. A chimeric antibody is a molecule in whichdifferent portions of the antibody are derived from different animalspecies, such as antibodies having a variable region derived from amurine monoclonal antibody and a human immunoglobulin constant region.Methods for producing chimeric antibodies are known in the art. Seee.g., Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214(1986); Gillies et al., (1989) J. Immunol. Methods 125:191-202; U.S.Pat. Nos. 5,807,715; 4,816,567; and 4,816,397, which are incorporatedherein by reference in their entirety.

Humanized antibodies are antibody molecules generated in a non-humanspecies that bind the desired antigen having one or more complementaritydetermining regions (CDRs) from the non-human species and framework (FR)regions from a human immunoglobulin molecule. Often, framework residuesin the human framework regions will be substituted with thecorresponding residue from the CDR donor antibody to alter, preferablyimprove, antigen binding. These framework substitutions are identifiedby methods well known in the art, e.g., by modeling of the interactionof the CDR and framework residues to identify framework residuesimportant for antigen binding and sequence comparison to identifyunusual framework residues at particular positions. (See, e.g., Queen etal., U.S. Pat. No. 5,585,089; Riechmann et al., Nature 332:323 (1988),which are incorporated herein by reference in their entireties).Antibodies can be humanized using a variety of techniques known in theart including, for example, CDR-grafting (EP 239,400; PCT publication WO91/09967; U.S. Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneeringor resurfacing (EP 592,106; EP 519,596; Padlan, Molecular Immunology28(4/5):489-498 (1991); Studnicka et al., Protein Engineering7(6):805-814 (1994); Roguska. et al., PNAS 91:969-973 (1994)), and chainshuffling (U.S. Pat. No. 5,565,332).

Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source that is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed following the methods of Winter and co-workers(Jones et al., Nature 321:522-525 (1986); Reichmann et al., Nature,332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988), bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such “humanized” antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossible some FR residues are substituted from analogous sites in rodentantibodies.

Completely human antibodies are particularly desirable for therapeutictreatment of human patients. Human antibodies can be made by a varietyof methods known in the art including phage display methods describedabove using antibody libraries derived from human immunoglobulinsequences. See also, U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCTpublications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO96/34096, WO 96/33735, and WO 91/10741; each of which is incorporatedherein by reference in its entirety. The techniques of Cole, et al., andBoerder et al., are also available for the preparation of humanmonoclonal antibodies (Cole et al., Monoclonal Antibodies and CancerTherapy, Alan R. Riss, (1985); and Boerner et al., J. Immunol.,147(1):86-95, (1991)).

Human antibodies can also be single-domain antibodies having a VH or VLdomain that functions independently of any other variable domain. Theseantibodies are typically selected from antibody libraries expressed inphage. These antibodies and methods for isolating such antibodies aredescribed in U.S. Pat. Nos. 6,595,142; 6,248,516; and applicationsUS20040110941 and US20030130496 all of which are incorporated herein byreference.

Human antibodies can also be produced using transgenic mice which areincapable of expressing functional endogenous immunoglobulins, but whichcan express human immunoglobulin genes. For example, the human heavy andlight chain immunoglobulin gene complexes may be introduced randomly orby homologous recombination into mouse embryonic stem cells.Alternatively, the human variable region, constant region, and diversityregion may be introduced into mouse embryonic stem cells in addition tothe human heavy and light chain genes. The mouse heavy and light chainimmunoglobulin genes may be rendered non-functional separately orsimultaneously with the introduction of human immunoglobulin loci byhomologous recombination. In particular, homozygous deletion of the JHregion prevents endogenous antibody production. The modified embryonicstem cells are expanded and microinjected into blastocysts to producechimeric mice. The chimeric mice are then bred to produce homozygousoffspring which express human antibodies. The transgenic mice areimmunized in the normal fashion with a selected antigen, e.g., all or aportion of a polypeptide of the invention. Monoclonal antibodiesdirected against the antigen can be obtained from the immunized,transgenic mice using conventional hybridoma technology. The humanimmunoglobulin transgenes harbored by the transgenic mice rearrangeduring B cell differentiation, and subsequently undergo class switchingand somatic mutation. Thus, using such a technique, it is possible toproduce therapeutically useful IgG, IgA, IgM and IgE antibodies. For anoverview of this technology for producing human antibodies, see Lonbergand Huszar, Int. Rev. Immunol. 13:65-93 (1995). For a detaileddiscussion of this technology for producing human antibodies and humanmonoclonal antibodies and protocols for producing such antibodies, see,e.g., PCT publications WO 98/24893; WO 92/01047; WO 96/34096; WO96/33735; European Patent No. 0 598 877; U.S. Pat. Nos. 5,413,923;5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318;5,885,793; 5,916,771; and 5,939,598, which are incorporated by referenceherein in their entirety. In addition, companies such as Abgenix, Inc.(Freemont, Calif.), Genpharm (San Jose, Calif.), and Medarex, Inc.(Princeton, N.J.) can be engaged to provide human antibodies directedagainst a selected antigen using technology similar to that describedabove.

Also human MAbs could be made by immunizing mice transplanted with humanperipheral blood leukocytes, splenocytes or bone marrows (e.g., Triomatechniques of XTL). Completely human antibodies which recognize aselected epitope can be generated using a technique referred to as“guided selection.” In this approach a selected non-human monoclonalantibody, e.g., a mouse antibody, is used to guide the selection of acompletely human antibody recognizing the same epitope. (Jespers et al.,Bio/technology 12:899-903 (1988)).

Further, antibodies to the polypeptides of the invention can, in turn,be utilized to generate anti-idiotype antibodies that “mimic”polypeptides of the invention using techniques well known to thoseskilled in the art. (See, e.g., Greenspan & Bona, FASEB J. 7(5):437-444;(1989) and Nissinoff, J. Immunol. 147(8):2429-2438 (1991)). For example,antibodies which bind to and competitively inhibit polypeptidemultimerization and/or binding of a polypeptide of the invention to aligand can be used to generate anti-idiotypes that “mimic” thepolypeptide multimerization and/or binding domain and, as a consequence,bind to and neutralize polypeptide and/or its ligand. Such neutralizinganti-idiotypes or Fab fragments of such anti-idiotypes can be used intherapeutic regimens to neutralize polypeptide ligand. For example, suchanti-idiotypic antibodies can be used to bind a polypeptide of theinvention and/or to bind its ligands/receptors, and thereby block itsbiological activity.

The antibodies of the present invention may be bispecific antibodies.Bispecific antibodies are monoclonal, preferably human or humanized,antibodies that have binding specificities for at least two differentantigens. in the present invention, one of the binding specificities maybe directed towards Factor D, the other may be for any other antigen,and preferably for a cell-surface protein, receptor, receptor subunit,tissue-specific antigen, virally derived protein, virally encodedenvelope protein, bacterially derived protein, or bacterial surfaceprotein, etc. Bispsecific antibodies may also comprise two or moresingle-domain antibodies.

Methods for making bispecific antibodies are well known. Traditionally,the recombinant production of bispecific antibodies is based on theco-expression of two immunoglobulin heavy-chain/light-chain pairs, wherethe two heavy chains have different specificites (Milstein and Cuello,Nature, 305:537-539 (1983). Because of the random assortment ofimmunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of ten different antibody molecules, ofwhich only one has the correct bispecific structure. The purification ofthe correct molecule is usually accomplished by affinity chromatographysteps. Similar procedures are disclosed in WO 93/08829, published May13, 1993, and in Traunecker et al., EMBO J., 10:3655-3659 (1991).

Antibody variable domains with the desired binding specificities(antibody-antigen combining sites) can be fused to immunoglobulinconstant domain sequences. The fusion preferably is with animmunoglobulin heavy-chain constant domain, comprising at least part ofthe hinge, CH2, and CH3 regions. It may have the first heavy-chainconstant region (CH1) containing the site necessary for light-chainbinding present in at least one of the fusions. DNAs encoding theimmunoglobulin heavy-chain fusions and, if desired, the immunoglobulinlight chain, are inserted into separate expression vectors, and areco-transformed into a suitable host organism. For further details ofgenerating bispecific antibodies sec, for example Suresh et al., Meth.In Enzym., 121:210 (1986).

Heteroconjugate antibodies are also contemplated by the presentinvention. Heteroconjugate antibodies are composed of two covalentlyjoined antibodies. Such antibodies have, for example, been proposed totarget immune system cells to unwanted cells (U.S. Pat. No. 4,676,980).It is contemplated that the antibodies may be prepared in vitro usingknown methods in synthetic protein chemistry, including those involvingcross-linking agents. For example, immunotoxins may be constructed usinga disulfide exchange reaction or by forming a thioester bond. Examplesof suitable reagents for this purpose include iminothiolate andmethyl-4-mercaptobutyrimidate and those disclosed, for example, in U.S.Pat. No. 4,676,980. In addition, one can generate single-domainantibodies to IL-33. Examples of this technology have been described inWO9425591 for antibodies derived from Camelidae heavy chain Ig, as wellin US20030130496 describing the isolation of single domain fully humanantibodies from phage libraries.

Generation of Monoclonal Antibodies (MABS)

In one embodiment of the invention, monoclonal antibodies, such asanti-Factor D, can be raised by immunizing rodents (e.g., mice, rats,hamsters and guinea pigs) with either native Factor D purified fromhuman plasma or urine, or recombinant Factor D or its fragmentsexpressed by either eukaryotic or prokaryotic systems. Other animals canbe used for immunization, e.g., non-human primates, transgenic miceexpressing human immunoglobulins and severe combined immunodeficient(SCID) mice transplanted with human B lymphocytes. Hybridomas can begenerated by conventional procedures by fusing b lymphocytes from theimmunized animals with myeloma cells (e.g.. Sp2/0 and NS0), as describedby G. Köhler and C. Milstein (Nature, 1975: 256: 495-497).

In addition, monoclonal antibodies ca be generated by screening ofrecombinant single-chain Fv or Fab libraries from human B lymphocytes inphage-display systems. The specificity of the MAbs to a given antigencan be tested by enzyme linked immunosorbent assay (ELISA), Westernimmunoblotting, or other immunochemical techniques. The inhibitoryactivity of the antibodies on complement activation can be assessed byhemolytic assays using unsensitized rabbit or guinea pig red blood cells(RBCs) for the alternative pathway, and using sensitized chicken orsheep RBCs for the classical pathway. The hybridomas in the positivewells are cloned by limiting dilution. The antibodies are purified forcharacterization for specificity to the antigen, such as Factor D, bythe assays well known in the art.

One can also create single peptide chain binding molecules in which theheavy and light chain Fv regions are connected. Single chain antibodies(“ScFv”) and the method of their construction are described in U.S. Pat.No. 4,946,778. Alternatively, Fab can be constructed and expressed bysimilar means (M. J. Evans et al., J. Immunol. Meth., 1995; 184:123-138). All of the wholly and partially human antibodies are lessimmunogenic than wholly murine MAbs, and the fragments and single chainantibodies are also less immunogenic. All these types of antibodies aretherefore less likely to evoke an immune or allergic response.Consequently, they are better suited for in vivo administration inhumans than wholly animal antibodies, especially when repeated orlong-term administration is necessary. In addition, the smaller size ofthe antibody fragment may help improve tissue bioavailability, which maybe critical for better dose accumulation in acute disease indications.

In one preferred embodiment of the invention, a chimeric Fab, havinganimal (mouse) variable regions and human constant regions is usedtherapeutically. The Fab is preferred because it is smaller than a wholeimmunoglobulin and may provide better tissue permeation; as monovalentmolecule, there is less chance of immunocomplexes and aggregatesforming; nd it can be produced in a micorbial system, which can moreeasily be sealed-up than a mammalian system.

Applications of the Complement Pathway Inhibitors

The complement inhibitors, such as antibodies and their bindingfragments, can be administered to subjects in an appropriatepharmaceutical formulation by a variety of routes, including, but notlimited, intravenous infusion, intravenous bolus injection, andintraperitoneal, intradermal, intramuscular, subcutaneous, intranasal,intratracheal, intraspinal, intracranial, and oral routes. Suchadministration enables them to bind to endogenous antigen, such asFactor D and thus inhibit the generation of C3b, C3a and C5aanaphylatoxins, and C5b-9.

The estimated preferred dosage of such antibodies and molecules isbetween 10 and 500 μg/ml of serum. The actual dosage can be determinedin clinical trials following the conventional methodology fordetermining optimal dosages, i.e., administering various dosages anddetermining which is most effective.

The complement pathway inhibitors can function to inhibit in vivocomplement activation and/or the alternative complement pathway andinflammatory manifestations that accompany it, such as recruitment andactivation of macrophages, neutrophils, platelets, and mast cells,edema, and tissue damage. These inhibitors can be used for treatment ofdiseases or conditions that are mediated by excessive or uncontrolledactivation of the complement system.

The antibodies of the present invention may be monospecific, bispecific,trispecific or of greater multispecificity. Multispecific antibodies maybe specific for different epitopes of a chosen antigen or may bespecific for both the antigen as well as for a heterologous epitope,such as a heterologous polypeptide or solid support material. See, e.g.,PCT publications WO 93/17715; WO 92/08802; WO 91/00360; WO 92/05793;Tutt, et al., J. Immunol. 147:60-69 (1991); U.S. Pat. Nos. 4,474,893;4,714,681; 4,925,648; 5,573,920; 5,601,819; Kostelny et al., J. Immunol.148:1547-1553 (1992).

Antibodies useful in the present invention may be described or specifiedin terms of the epitope(s) or portion(s) of a complement pathwaycomponent, such as Factor D, which they recognize or specifically bind.The epitope(s) or polypeptide portion(s) may be specified as describedherein, e.g., by N-terminal and C-terminal positions, by size incontiguous amino acid residues.

Antibodies useful in the present invention may also be described orspecified in terms of their cross-reactivity. Antibodies the bindcomplement pathway component polypeptides, which have at least 95%, atleast 90%, at least 85%, at least 80%, at least 75%, at least 70%, atleast 65%, at least 60%, at least 55%, and at least 50% identity (ascalculated using methods known in the art and described herein) to IL-13are also included in the present invention. Anti-Factor D antibodies mayalso bind with a KD of less than about 10-7 M, less than about 10-6 M,or less than about 10-5 M to other proteins, such as Factor D antibodiesfrom species other than that against which the anti-Factor D antibody isdirected.

Vectors and Host Cells

In another aspect, the present invention provides vector constructscomprising a nucleotide sequence encoding the antibodies of the presentinvention and a host cell comprising such a vector. Standard techniquesfor cloning and transformation may be used in the preparation of celllines expressing the antibodies of the present invention.

Recombinant expression vectors containing a nucleotide sequence encodingthe antibodies of the present invention can be prepared using well knowntechniques. The expression vectors include a nucleotide sequenceoperably linked to suitable transcriptional or translational regulatorynucleotide sequences such as those derived from mammalian, microbial,viral, or insect genes. Examples of regulatory sequences includetranscriptional promoters, operators, enhancers, mRNA ribosomal bindingsites, and/or other appropriate sequences which control transcriptionand translation initiation and termination. Nucleotide sequences are“operably linked” when the regulatory sequences functionally relates tothe nucleotide sequence of the appropriate polypeptide. Thus, a promotornucleotide sequence is operably linked to, e.g., the antibody heavychain sequence if the promoter nucleotide sequence controls thetranscription of the appropriate nucleotide sequence.

In addition, sequences encoding appropriate signal peptides that are notnaturally associated with antibody heavy an/or light chain sequences canbe incorporated into expression vectors. For example, a nucleotidesequence for a signal peptide (secretory leader) may be fused in-frameto the polypeptide sequence so that the antibody is secreted to theperiplasmic space or into the medium. A signal peptide that isfunctional in the intended host cells enhances extracellular secretionof the appropriate antibody. The signal peptide may be cleaved from thepolypeptide upon secretion of antibody from the cell. Examples of suchsecretory signals are well known and include, e.g., those described inU.S. Pat. No. 5,698,435, U.S. Pat. No. 5,698,417, and U.S. Pat. No.6,204,023.

Host cells useful in the present invention include but are not limitedto microorganisms such as bacteria (e.g., E. coli, B. subtilis)transformed with recombinant bacteriophage DNA, plasmid DNA or cosmidDNA expression vectors containing antibody coding sequences; yeast(e.g., Saccharomyces, Pichia) transformed with recombinant yeastexpression vectors containing antibody coding sequences; insect cellsystems infected with recombinant virus expression vectors (e.g.,Baculovirus) containing antibody coding sequences; plant cell systemsinfected with recombinant virus expression vectors (e.g., cauliflowermosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed withrecombinant plasmid expression vectors (e.g., Ti plasmid) containingantibody coding sequences; or mammalian cell systems (e.g., COS, CHO,BHK, 293, 3T3 cells) harboring recombinant expression constructcontaining promoters derived from the genome of mammalian cells (e.g.,metallothionein promoter) or from mammalian viruses (e.g., theadenovirus late promoter; the vaccinia virus 7.5K promoter).

The vector may be a plasmid vector, a single or double-stranded phagevector, or a single or double-stranded RNA or DNA viral vector. Suchvectors may be introduced into cells as polynucleotides by well knowntechniques for introducing DNA and RNA into cells. The vectors, in thecase of phage and viral vectors also may be introduced into cells aspackaged or encapsulated virus by well known techniques for infectionand transduction. Viral vectors may be replication competent orreplication defective. In the later case, viral propagation generallywill occur only in complementing host cells. Cell-free translationsystems may also be employed to produce the protein using RNAs derivedfrom the present DNA constructs. Such vectors may include the nucleotidesequence encoding the constant region of the antibody molecule (see,e.g., PCT Publication WO 86/05807; PCT Publication WO 89/01036; U.S.Pat. No. 5,122,464) and the variable domain of the antibody may becloned into such a vector for expression of the entire heavy or lightchain.

Prokaryotes useful as host cells in the present invention include gramnegative or gram positive organisms such as E. coli, and B. subtilis.Expression vectors for use in prokaryotic host cells generally compriseone or more phenotypic selectable marker genes. A phenotypic selectablemarker gene is, for example, a gene encoding a protein that confersantibiotic resistance or that supplies an autotrophic requirement.Examples of useful expression vectors for prokaryotic host cells includethose derived from commercially available plasmids such as the pKK223-3(Pharmacia Fine Chemicals, Uppsala, Sweden) pGEM1 (Promega Biotec,Madison, Wis., USA), and the pET (Novagen, Madison, Wis., USA) and pRSET(Invitrogen Corporation, Carlsbad, Calif., USA) series of vectors(Studier, F. W., J. Mol. Biol. 219: 37 (1991); Schoepfer, R. Gene 124:83 (1993)). promoter sequences commonly used for recombinant prokaryotichost cell expression vectors include T7, (Rosenberg, et al. Gene 56,125-135 (1987)), β-lactamase (penicillinase), lactose promoter system(Chang et al., Nature 275:615, (1987); and Goeddel et al., Nature281:544, (1979)), tryptophan (trp) promoter system (Goeddel et al.,Nucl. Acids Res. 8:4057, (1980)), and tac promotor (Sambrook et al.,1980, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y.)

Yeasts useful in the present invention include those from the genusSaccharomyces, Pichia, Actinomycetes and Kluyveromyces. Yeast vectorswill often contain an origin of replication sequence from a 2μ yeastplasmid, an autonomously replicating sequence (ARS), a promoter region,sequences for polyadenylation, sequences for transcription termination,and a selectable marker gene. Suitable promoter sequences for yeastvectors include, among others, promoters for methallothionein,3-phosphoglycerate kinase (Hitzeman et al., J. Biol. Chem. 255:2073,(19080)) or other glycolytic enzymes (Holland et al., Biochem. 17:4900,(1978)) such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase. Other suitable vectors and promoters for use in yeastexpression are further described in Fleer et al., Gene, 107:285-195(1991). Other suitable promoters and vectors for yeast and yeasttransformation protocols are well known in the art. Yeast transformationprotocols are well known. One such protocol is described by Hinnen etal., Proc. Natl. Acad. Sci., 75:1929 (1978). The Hinnen protocol selectsfor Trp+ transformants in a selective medium.

Mammalian or insect host cell culture systems may also be employed toexpress recombinant antibodies, e.g., Baculovirus systems for productionof heterologous proteins. In an insect system, Antographa californicanuclear polyhedrosis virus (AcNPV) may be used as a vector to expressforeign genes. The virus grows in Spodoptera frugiperda cells. Theantibody coding sequence may be cloned individually into non-essentialregions (for example the polyhedrin gene) of the virus and placed undercontrol of an AcNPV promotor (for example the polyhedrin promoter).

NSD or Chinese hamster ovary (CHO) cells for mammalian expression of theantibodies of the present invention may be used. Transcriptional andtranslational control sequences for mammalian host cell expressionvectors may be excised from viral genomes. Commonly used promotersequences and enhancer sequences are derived from Polyoma virus,Adenovirus 2, Simian Virus 40 (SV40), and human cytomegalovirus (CMV).DNA sequences derived from the SV40 viral genome may be used to provideother genetic elements for expression of a structural gene sequence in amammalian host cell, e.g., SV40 origin, early and late promoter,enhancer, splice, and polyadenylation sites. Viral early and latepromoters are particularly useful because both are easily obtained froma viral genome as a fragment which may also contain a viral origin ofreplication. Exemplary expression vectors for use in mammalian hostcells are commercially available.

Polynucleotides Encoding Antibodies

The invention further provides polynucleotides or nucleic acids, e.g.,DNA, comprising a nucleotide sequence encoding an antibody of theinvention and fragments thereof. Exemplary polynucleotides include thoseencoding antibody chains comprising one or more of the amino acidsequences described herein. The invention also encompassespolynucleotides that hybridize under stringent or lower stringencyhybridization conditions to polynucleotides that encode an antibody ofthe present invention.

The polynucleotides may be obtained, and the nucleotide sequence of thepolynucleotides determined, by any method known in the art. For example,if the nucleotide sequence of the antibody is known, a polynucleotideencoding the antibody may be assembled from chemically synthesizedoligonucleotides (e.g., as described in Kutmeier et al., BioTechniques17:242 (1994)), which, briefly, involves the synthesis of overlappingoligonucleotides containing portions of the sequence encoding theantibody, annealing and ligating of those oligonucleotides, and thenamplification of the ligated oligonucleotides by PCR.

Alternatively, a polynucleotide encoding an antibody may be generatedfrom nucleic acid from a suitable source. If a clone containing anucleic acid encoding a particular antibody is not available, but thesequences of the antibody molecule is known, a nucleic acid encoding theimmunoglobulin may be chemically synthesized or obtained from a suitablesource (e.g., an antibody cDNA library, or a cDNA library generatedfrom, or nucleic acid, preferably poly A+ RNA, isolated from, any tissueor cells expressing the antibody, such as hybridoma cells selected toexpress an antibody of the invention) by PCR amplification usingsynthetic primers hybridizable to the 3′ and 5′ ends of the sequence orby cloning using an oligonucleotide probe specific for the particulargene sequence to identify, e.g., a cDNA clone from a cDNA library thatencodes the antibody. Amplified nucleic acids generated by PCR may thenbe cloned into replicable cloning vectors using any method well known inthe art.

Once the nucleotide sequence and corresponding amino acid sequence ofthe antibody is determined, the nucleotide sequence of the antibody maybe manipulated using methods well known in the art for the manipulationof nucleotide sequences, e.g., recombinant DNA techniques, site directedmutagenesis, PCR, etc. (see, for example, the techniques described inSambrook et al., 1990, Molecular Cloning, A Laboratory Manual, 2d Ed.,Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. and Ausubel etal., eds., 1998, Current Protocols in Molecular Biology, John Wiley andSons, N.Y., which are both incorporated by reference herein in theirentireties), to generate antibodies having a different amino acidsequence, for example to create amino acid substitutions, deletions,and/or insertions.

In a specific embodiment, the amino acid sequence of the heavy and/orlight chain variable domains may be inspected to identify the sequencesof the CDRs by well known methods, e.g., by comparison to known aminoacid sequences of other heavy and light chain variable regions todetermine the regions of sequence hypervariability. Using routinerecombinant DNA techniques, one or more of the CDRs may be insertedwithin framework regions, e.g., into human framework regions to humanizea non-human antibody, as described supra. The framework regions may benaturally occurring or consensus framework regions, and preferably humanframework regions (see, e.g., Chothia et al., J. Mol. Biol. 278: 457-479(1998) for a listing of human framework regions). Preferably, thepolynucleotide generated by the combination of the framework regions andCDRs encodes an antibody that specifically binds a polypeptide of theinvention. Preferably, as discussed supra, one or more amino acidsubstitutions may be made within the framework regions, and, preferably,the amino acid substations improve binding of the antibody to itsantigen. Additionally, such methods may be used to make amino acidsubstitutions or deletions of one or more variable region cysteineresidues participating in an intrachain disulfide bond to generateantibody molecules lacking one or more intrachain disulfide bonds. Otheralterations to the polynucleotide are encompassed by the presentinvention and within the skill of the art.

In addition, techniques developed for the production of “chimericantibodies” (Morrison et al., Proc Natl. Acad. Sci. 81:851-855 (1984);Neuberger et al., Nature 312:604-608 (19840; Takeda et al., Nature314:452-454 (1985)) by splicing genes from a mouse antibody molecule ofappropriate antigen specificity together with genes from a humanantibody molecule of appropriate biological activity can be used. Asdescribed supra, a chimeric antibody is a molecule in which differentportions are derived from different animal species, such as those havinga variable region derived from a murine MAb and a human immunoglobulinconstant region, e.g., humanized antibodies.

Alternatively, techniques described for the production of single chainantibodies (U.S. Pat. No. 4,946,778; Bird, Science 242:423-42 (1988);Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Wardet al., Nature 334:544-54 (1989)) can be adapted to produce single chainantibodies. Single chain antibodies are formed by linking the heavy andlight chain fragments of the Fv region via an amino acid bridge,resulting in a single chain polypeptide. Techniques for the assembly offunctional Fv fragments in E. coli may also be used (Skerra et al.,Science 242:1038-1041 (1988)).

Methods of Producing Antibodies

The antibodies of the invention can be produced by any method known inthe art for the synthesis of antibodies, in particular, by chemicalsynthesis or preferably, by recombiant expression techniques.

Recombinant expression of an antibody of the invention, or fragment,derivative or analog thereof, (e.g., a heavy or light chain of anantibody of the invention or a single chain antibody of the invention),requires construction of an expression vector containing apolynucleotide that encodes the antibody or a fragment of the antibody.Once a polynucleotide encoding an antibody molecule has been obtained,the vector for the production of the antibody may be produced byrecombinant DNA technology. An expression vector is constructedcontaining antibody coding sequences and appropriate transcriptional andtranslational control signals. These methods include, for example, invitro recombinant DNA techniques, synthetic techniques, and in vivogenetic recombination.

The expression vector is transferred to a host cell by conventionaltechniques and the transfected cells are then cultured by conventionaltechniques to produce an antibody of the invention. In one aspect of theinvention, vectors encoding both the heavy and light chains may beco-expressed in the host cell for expression of the entireimmunoglobulin molecule, as detailed below.

A variety of host-expression vector system may be utilized to expressthe antibody molecules of the invention as described above. Suchhost-expression systems represent vehicles by which the coding sequencesof interest may be produced and subsequently purified, but alsorepresent cells which may, when transformed or transacted with theappropriate nucleotide coding sequences, express an antibody molecule ofthe invention in situ. Bacterial cells such as E. coli, and eukaryoticcells are commonly used for the expression of a recombinant antibodymolecule, especially for the expression of whole recombinant antibodymolecule. For example, mammalian cells such as Chinese hamster ovarycells (CHO), in conjunction with a vector such as the major intermediateearly gene promoter element from human cytomegalovirus is an effectiveexpression system for antibodies (Foecking et al., Gene 45:101 (1986);Cockett et al., Bio/Technology 8:2 (1990)).

In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modification (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed. To thisend, eukaryotic host cells which possess the cellular machinery forproper processing of the primary transcript, glycosylation, andphosphorylation of the gene product may be used. Such mammalian hostcells include, but are not limited to, CHO, COS, 293, 3T3, or myelomacells.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressthe antibody molecule may be engineered. Rather than using expressionvectors which contain viral origins of replication, host cells can betransformed with DNA controlled by appropriate expression controlelements; (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker its the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci which in turncan be cloned and expanded into cell lines. This method mayadvantageously be used to engineer cell lines which express the antibodymolecule. Such engineered cell lines may be particularly useful inscreening and evaluation of compounds that interact directly orindirectly with the antibody molecule.

A number of selection systems, may be used, including but not limited tothe herpes simplex virus thymidine kinase (Wigler et al., Cell 11:273(1977), hypoxanthine-guanine phosphoribosyltransferase (Szybalska &Szybalski, Proc. Natl. Acad. Sci. USA 48:202 (1992)), and adeninephosphorihosytransferase (Lowy et al., Cell 22:817 (1980)) genes can beemployed in tk, hgprt or aprt-cells, respectively. Also, antimetaboliteresistance can be used as the basis of selection for the followinggenes: dhfr, which confers resistance to methotrexate (Wigler et al.,Proc. Natl. Acad. Sci., USA 77:357 (1980); O'Hare et al., Proc. Natl.Acad. Sci. USA 78:1527 (1981)); gpt, which confers resistance tomycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78:2072(1981)); neo, which confers resistance to the aminoglycoside G-418 (Wuand Wu, Biotherapy 3:87-95 (1991)); and hygro, which confers resistanceto hygromycin (Santerre et al., Gene 30:147 (1984)). Methods commonlyknown in the art of recombinant DNA technology may be routinely appliedto select the desired recombinant clone, and such methods are described,for example, in Ausubel et al. (eds.), Current Protocols in MolecularBiology, John Wiley & Sons, NY (1993); Kriegler, Gene Transfer andExpression, A Laboratory Manual, Stockton Press, NY (1980); and inChapters 12 and 13, Dracopoli et al. (eds), Current Protocols in HumanGenetics, John Wiley & Sons, NY (1994); Colberre-Garapin et al., J. Mol.Biol. 150:1 (1981), which are incorporated by reference herein in theirentireties.

The expression levels of an antibody molecule can be increased by vectoramplification (for a review, see Bebbington and Hentschel, “The use ofvectors based on gene amplification for the expression of cloned genesin mammalian cells” (DNA Cloning, Vol. 3. Academic Press, New York,1987)). When a marker in the vector system expressing antibody isamplifiable, increase in the level of inhibitor present in culture ofhost cell will increase the number of copies of the marker gene. Sincethe amplified region is associated with the antibody gene, production ofthe antibody will also increase (Crouse et al., Mol. Cell. Biol. 3:257(1983)).

The host cell may be co-transfected with two expression vectors of theinvention, the first vector encoding a heavy chain derived polypeptideand the second vector encoding a light chain derived polypeptide. Thetwo vectors may contain identical selectable markers which enable equalexpression of heavy and light chain polypeptides. Alternatively, asingle vector may be used which encodes, and is capable of expressing,both heavy and light chain polypeptides. In such situations, the lightchain should be placed before the heavy chain to avoid an excess oftoxic free heavy chain (Proudfoot, Nature 322:52 (1986); Kohler, Proc.Natl. Acad. Sci. USA 77:2197 (1980)). The coding sequences for the heavyand light chains may comprise cDNA or genomic DNA.

Once an antibody molecule of the invention has been produced by ananimal, chemically synthesized, or recombinantly expressed, it may bepurified by any method known in the art for purification of animmunoglobulin molecule, for example, by chromatography (e.g., ionexchange, affinity, particularly by affinity for the specific antigenafter protein A, and size-exclusion chromatography), centrifugation,differential solubility, or by any other standard technique for thepurification of proteins. In addition, the antibodies of the presentinvention or fragments thereof can be fused to heterologous polypeptidesequences described herein or otherwise known in the art, to facilitatepurification.

The present invention encompasses antibodies recombinantly fused orchemically conjugated (including both convalently and non-covalentlyconjugations) to a polypeptide. Fused or conjugated antibodies of thepresent invention may be used for ease in purification. See e.g., Harboret al., supra, and PCT publication WO 93/21232; EP 439,095; Naramura etal., Immunol. Lett. 39:91-99 (1994); U.S. Pat. No. 5,474,981; Gillies etal., Proc. Natl. Acad. Sci. 89:1328-1432. (19920; Fell et al., J.Immunol. 146:2446-2452 (1991), which are incorporated by reference intheir entireties.

Moreover, the antibodies or fragments thereof of the present inventioncan be fused to marker sequences, such as a peptide to facilitatepurification. In preferred embodiments, the marker amino acid sequenceis a hexa-histidine peptide, such as the tag provided in a pQE vector(QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), amongother, many of which are commercially available. As described in Gentzet al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance,hexa-histidine provides for convenient purification of the fusionprotein. Other peptide tags useful for purification include, but are notlimited to, the “HA” tag, which corresponds to an epitope derived fromthe influenza hemagglutinin protein (Wilson et al., Cell 37:767 (1984))and the “flag” tag.

Diagnostic Uses for Antibodies

The antibodies of the invention include derivatives that are modified,i.e., by the covalent attachment of any type of molecule to theantibody, such that covalent attachment does not interfere with bindingto the antigen. For example, but not by way of limitation, the antibodyderivatives include antibodies that have been modified, e.g., bybiotinylation, HRP, or any other detectable moiety.

Antibodies of the present invention may be used, for example, but notlimited to, to detect Factor D, including both in vitro and in vivodiagnostic methods. For example, the antibodies have use in immunoassaysfor qualitatively and quantitatively measuring levels of Factor D inbiological samples obtained from the eyes of subjects suffering fromocular conditions or diseases. Typically immunoassays are described in,e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold SpringHarbor Laboratory Press, 2nd ed. 1988) (incorporated by reference hereinin its entirety).

As discussed in more detail below, the antibodies of the presentinvention may be used either alone or in combination with othercompositions. The antibodies may further be recombinantly fused to aheterologous polypeptide at the N- or C-terminus or chemicallyconjugated (including covalently and non-covalently conjugations) topolypeptides or other compositions. For example, antibodies of thepresent invention may be recombinantly fused or conjugated to moleculesuseful as labels in detection assays.

The present invention further encompasses the use of antibodies orfragments thereof conjugated to a diagnostic agent for the detection ofthe levels of complement pathway components in the eye of an affectedindividual. The antibodies can be used diagnostically to, for example,monitor the development or progression of an ocular condition or diseaseas part of a clinical testing procedure to, e.g., determine the efficacyof a given treatment regimen. Detection can be facilitated by couplingthe antibody to a detectable substance. Examples of detectablesubstances include various enzymes, prosthetic groups, fluorescentmaterials, luminescent materials, bioluminescent materials, radioactivematerials, positron emitting metals using various positron emissiontomographies, and nonradioactive paramagnetic metal ions. The detectablesubstance may be coupled or conjugated either directly to the antibody(or fragment thereof) or indirectly, through an intermediate (such as,for example, a linker known in the art) using techniques known in theart. See, for example, U.S. Pat. No. 4,741,900 for metal ions which canbe conjugated to antibodies for use as diagnostics according to thepresent invention. Examples of suitable enzymes include horseradishperoxidase, alkaline phosphatase, beta-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyante, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin; and examples of suitable radioactive materialinclude 125I, 131I, 111In or −99Tc.

Antibodies may also be attached to solid supports, which areparticularly useful for immunoassays or purification of the targetantigen. Such solid supports include, but are not limited to, glass,cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride orpolypropylene.

Labeled antibodies, and derivatives and analogs thereof, whichspecifically bind to Factor D can be used for diagnostic purposes todetect, diagnose, or monitor diseases, disorders, and/or conditionsassociated with the aberrant expression and/or activity of Factor D. Theinvention provides for the detection of aberrant expression of Factor D,comprising (a) assaying the expression of Factor D in cells or bodyfluid of an individual using one or more antibodies of the presentinvention specific to Factor D and (b) comparing the level of geneexpression with a standard gene expression level, whereby an increase ordecrease in the assayed Factor D expression level compared to thestandard expression level is indicative of aberrant expression.

Antibodies may be used for detecting the presence and/or levels ofFactor D in a sample, e.g., ocular fluid. The detecting method maycomprise contacting the sample with an anti-Factor D antibody anddetermining the amount of antibody that is bound to the sample.

The invention provides a diagnostic assay for diagnosing a disorder,comprising (a) assaying the expression of Factor D in cells or bodyfluid of an individual using one or more antibodies of the presentinvention and (b) comparing the level of gene expression with a standardgene expression level, whereby an increase or decrease in the assayedgene expression level compared to the standard expression level isindicative of a particular disorder.

Antibodies of the invention can be used to assay protein levels in abiological sample using classical immunohistological methods known tothose of skill in the art (e.g., see Jalkanen, et al., J. Cell. Biol.101:976-985 (1985); Jalkanen, et al., J. Cell. Bio. 105:3087-3096(1987)). Other antibody-based methods useful for detecting protein geneexpression include immunoassays, such as the enzyme linked immunosorbentassay (ELISA) and the radioimmunoassay (RIA). Suitable antibody assaylabels are known in the art and include enzyme labels, such as, glucoseoxidase; radioisotopes, such as iodine (125I, 121I), carbon (14C),sulfur (35S), tritium (3H), indium (112In), and technetium (99Tc);luminescent labels, such as luminol; and fluorescent labels, such asfluorescein and rhodamine, and biotin.

One aspect of the invention is the detection and diagnosis of a diseaseor disorder associated with complement activation in the eyes of asubject, preferably a mammal and most preferably a human. In oneembodiment, diagnosis comprises: a) taking a sample from the eye of apatient; b) measuring the level of complement components, such as C3a orC3b or C5a. Background level can be determined by various methodsincluding, comparing the amount of labeled molecule detected to astandard value previously determined for a particular system.

In an embodiment, monitoring of the disease or disorder is carried outby repeating the method for diagnosing the disease or disease, forexample, one month after initial diagnosis, six months after initialdiagnosis, one year after initial diagnosis, etc.

Therapeutic Uses of Complement Pathway Inhibitors

Complement pathway inhibitors may be administered to a subject sufferingfrom an ocular disease such as age-related mascular degeneration. Anantibody, with or without a therapeutic moiety conjugated to it, can beused as a therapeutic. The present invention is directed to the use ofcomplement pathway inhibitors, particularly antibodies, comprisingadministering said inhibitors to an animal, a mammal, or a human, fortreating a ocular disease, disorder, or condition involving complementpathway activation. The animal or subject may be an animal in need of aparticular treatment, such as an animal having been diagnosed with aparticular disorder, e.g., one relating to complement. Antibodiesdirected against Factor D are useful for inhibiting the alternativecomplement pathway and thus inhibiting complement pathway relateddisorder or conditions. In particular, the present invention relates tothe treatment of AMD, diabetic retinopathy, and choroidalneovascularization. For example, by administering a therapeuticallyacceptable dose of an antibody, or antibodies, of the present invention,or a cocktail of the present antibodies, or in combination with othermolecules of varying sources, the effects of activation of complementpathway components may be reduced or eliminated in the treated mammal.

Therapeutic compounds of the invention include, but are not limited to,antibodies of the invention (including fragments, analogs andderivatives thereof as described herein)and nucleic acids encodingantibodies of the invention as described below (including fragments,analogs and derivatives thereof and anti-idiotypic antibodies asdescribed herein). The antibodies of the invention can be used to treat,inhibit or prevent diseases, disorders or conditions associated withaberrant expression and/or activity of the complement pathway,particularly the alternative pathway, and particularly Factor D. Thetreatment and/or prevention of diseases, disorders, or conditionsassociated with aberrant expression and/or activity of Factor Dincludes, but is not limited to, alleviating at least one symptomsassociated with those diseases, disorders or conditions. Antibodies ofthe invention may be provided in pharmaceutically acceptablecompositions as known in the art or as described herein.

The amount of the antibody which will be effective in the treatment,inhibition and prevention of a disease or disorder associated withaberrant expression and/or activation of the complement pathway can bedetermined by standard clinical techniques. The antibody can beadministered in treatment regimes consistent with the disease, e.g., asingle or a few doses over one to several days to ameliorate a diseasestate or periodic doses over an extended time to prevent ocular diseasesor conditions.

In addition, in vitro assays may optionally be employed to help identifyoptimal dosage ranges. The precise dose to be employed in theformulation will also depend on the route of administration, and theseriousness of the disease or disorder, and should be decided accordingto the judgement of the practitioner and each patient's circumstances.Effective doses may be extrapolated from dose-response curves derivedfrom in vitro or animal model test systems.

For antibodies, the dosage administered to a patient is typically 0.1mg/kg to 100 mg/kg of the patient's body weight. Preferably, the dosageadministered to a patient is between 0.1 mg/kg and 20 mg/kg of thepatient's body weight, more preferably 1 mg/kg to 10 mg/kg of thepatient's body weight. Generally, human antibodies have a longerhalf-life within the human body than antibodies from other species dueto the immune response to the foreign polypeptides. Thus, lower dosagesof human antibodies and less frequent administration is often possible.Further, the dosage and frequency of administration of antibodies of theinvention may be reduced by enhancing uptake and tissue penetration(e.g., into the brain) of the antibodies by modifications such as, forexample, lipidation. In a preferred aspect, the antibody issubstantially purified (e.g., substantially free from substances thatlimit its effect or produce undesired side-effects).

Various delivery systems are known and can be used to administer anantibody of the present invention, including injection, e.g.,encapsulation in liposomes, microparticles, microcapsules, recombinantcells capable of expressing the compound, receptor-mediated endocytosis(see, e.g., Wu et al., J. Biol. Chem. 262:4429-4432 (1987)),construction of a nucleic acid as part of a retroviral or other vector,etc.

The antibody can be administered to the mammal in any acceptable manner.Methods of introduction include but are not limited to intradermal,intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal,epidural, inhalation and oral routes. However, for purpose of thepresent invention, the preferred route of administration is intraocular.

Administration can be systemic or local. in addition, it may bedesirable to introduce the therapeutic antibodies or compositions of theinvention into the central nervous system by any suitable route,including intraventricular and intrathecal injection; intraventricularinjection may be facilitated by an intraventricular catheter, forexample, attached to a reservoir, such as an Ommaya reservoir.

In another embodiment, the antibody can be delivered in a vesicle, inparticular a liposome (see Langer, Science 249:1527-1533 (1990); Treatel al., in Liposomes in the Therapy of Infectious Disease and Cancer,Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989);Lopez-Berestein, ibid., pp. 317-327; see generally ibid.).

In yet another embodiment, the antibody can be delivered in a controlledrelease system. In one embodiment, a pump may be used (see Langer,supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald etal., Surgery 88:507 (1980); Saudek et al., N. Engl. J. med. 321:574(1989)). In another embodiment, polymeric materials can be used (seeMedical Applications of Controlled Release, Langer and Wise (eds.), CRCPres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability, DrugProduct Design and Performance, Smolen and Ball (eds.), Wiley, New York(1984); Ranger and Peppas, J., Macromol. Sci. Rev. Macromol. Chem. 23:61(1983); see also Levy et al., Science 228:190 (1985); During et al.,Ann. Neurol. 25:351 (1989); Howard et al., J. Neurosurg. 71:105 (1989)).In yet another embodiment, a controlled release system can be placed inproximity of the therapeutic target.

The present invention also provides pharmaceutical compositions usefulin the present method. Such compositions comprise a therapeuticallyeffective amount of the antibody, and a physiologically acceptablecarrier. In a specific embodiment, the term “physiologically acceptable”means approved by a regulatory agency of the Federal or a stategovernment or listed in the U.S. Pharmacopeia or other generallyrecognized pharmacopeia for use in animals, and more particularly inhumans. The term “carrier” refers to a diluent, adjuvant, excipient, orvehicle with which the therapeutic is administered. Such physiologicalcarriers can be sterile liquids, such as water and oils, including thoseof petroleum, animal, vegetable or synthetic origin, such as peanut oil,soybean oil, mineral oil, sesame oil and the like. Water is a preferredcarrier when the pharmaceutical composition is administeredintravenously. Saline solutions and aqueous dextrose and glycerolsolutions can also be employed as liquid carriers, particularly forinjectable solutions. Suitable pharmaceutical excipients include starch,glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silicagel, sodium stearate, glycerol monostearate, talc, sodium chloride,dried skim milk, glycerol, propylene, glycol, water, ethanol and thelike. The composition, if desired, can also contain minor amounts ofwetting or emulsifying agents, or pH buffering agents. Thesecompositions can take the form of solutions, suspensions, emulsion,tablets, pills, capsules, powders, sustained release formulations andthe like. The composition can be formulated as a suppository, withtraditional binders and carriers such as triglycerides. oral formulationcan include standard carriers such as pharmaceutical grades of mannitol,lactose, starch, magnesium stearate, sodium saccharine, cellulose,magnesium carbonate, etc. Examples of suitable carriers are described in“Remington's Pharmaceutical Sciences” by E. W. Martin. Such compositionswill contain an effective amount of the antibody, preferably in purifiedform, together with a suitable amount of carrier so as to provide theform for proper administration to the patient. The formulation shouldsuit the mode of administration.

In one embodiment, the composition is formulated in accordance withroutine procedures as a pharmaceutical composition adapted forintravenous administration to human beings. Typically, compositions forintravenous administration are solutions in sterile isotonic aqueousbuffer. Where necessary, the composition may also include a solubilizingagent and a local anesthetic such as lignocaine to ease pain at the siteof the injection. Generally, the ingredients are supplied eitherseparately or mixed together in unit dosage form, for example, as a drylyophilized powder or water free concentrate in a hermetically sealedcontainer such as an ampoule or sachette indicating the quantity ofactive agent. Where the composition is to be administered by infusion,it can be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the composition isadministered by injection, an ampoule of sterile water for injection orsaline can be provided so that the ingredients may be mixed prior toadministration.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Optionally associated withsuch container(s) can be a notice in the form prescribed by agovernmental agency regulating the manufacture, use of sale ofpharmaceuticals or biological products, which notice reflects approvalby the agency of manufacture, use of sale for human administration.

In addition, the antibodies of the present invention may be conjugatedto various effector molecules such as heterologous polypeptides, drugs,radionucleotides, or toxins. See, e.g., PCT publications WO 92/08495; WO91/14438; WO 89/12624; U.S. Pat. No. 5,314,995; and EP 396,387. Anantibody or fragment thereof may be conjugated to a therapeutic moietysuch as a cytotoxin, e.g., a cytostatic or cytocidal agent, atherapeutic agent or a radioactive metal ion, e.g., alpha-emitters suchas, for example, 213Bi. A cytotoxin or cytotoxic agent includes anyagent that is detrimental to cells. Examples include paclitaxol,cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin,etoposide, tneoposide, bincristine, binblastine, colchicin, doxorubicin,daunorubicin, dihydorxy anthracin dione, mitoxantrone, mithramycin,actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,tetracaine, lidocaine, propranolol, and puromycin and analogs orhomologues thereof. Therapeutic agents include, but are not limited to,antimetabolites (e.g., methotrexate, 6-mercapiopurine, 6-thioguanine,cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g.,mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) andlomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine).

Techniques for conjugating such therapeutic moiety to antibodies arewell known, see, e.g., Arnon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”,Immunol. Rev. 62:119-58 (1982). Alternatively, an antibody can beconjugated to a second antibody to form an antibody heterconjugate.(See, e.g., Segal in U.S. Pat. No. 4,676,980.)

The conjugates of the invention can be used for modifying a givenbiological response, the therapeutic agent or drug moiety is not to beconstrued as limited to classical chemical therapeutic agents. Forexample, the drug moiety may be a protein or polypeptide possessing adesired biological activity. Such proteins may include, for example, atoxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin;a protein such as tumor necrosis Factor, α-interferon, β-interferon,nerve growth Factor, platelet derived growth Factor, tissue plasminogenactivator, an apoptotic agent, e.g., TNF-α, TNF-β, AIM I (See,International Publication No. WO 97/33899), AIM II (See, InternationalPublication No. WO 97/34911), Fas Ligand (Takahashi et al., Int.Immunol., 6:1567-1574 (1994)), VEGI (See, International Publication No.WO 99/23105), a thrombotic agent or an anti-angiogenic agent, e.g.,angiostatin or endostatin; or, biological response modifiers such as,for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2(“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colonystimulating Factor (“GM-CSF”), granulocyte colony stimulating Factor(“G-CSF”), or other growth Factors.

Antibody-Based Gene Therapy

In a another aspect of the invention, nucleic acids comprising sequencesencoding antibodies or binding fragments thereof, are administered totreat, inhibit or prevent a disease or disorder associated with aberrantexpression and/or activation of the complement pathway by way of genetherapy. Gene therapy refers to therapy performed by the administrationto a subject of an expressed or expressible nucleic acid. In thisembodiment of the invention, the nucleic acids produce their encodedprotein that mediates a therapeutic effect. Any of the methods for genetherapy available can be used according to the present invention.Exemplary methods are described below.

For general reviews of the methods of gene therapy, see Goldspiel etal., Clinical Pharmacy 12:488-505 (1983); Wu and Wu, Biotherapy 3:87-95(1991); Tolstoshev, Ann. Rev. Pharmacol, Toxicol, 32:573-596 (19930;Mulligan, Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev.Biochem. 62:191-217 (1993); May, TIBTECH 11(5):155-215 (1993).

In a one aspect, the compound comprises nucleic acid sequences encodingan antibody, said nucleic acid sequences being part of expressionvectors that express the antibody or fragments or chimeric proteins orheavy or light chains thereof in a suitable host. In particular, suchnucleic acid sequences have promoters operably linked to the antibodycoding region, said promoter being inducible or constitutive, and,optionally, tissue-specific.

In another particular embodiment, nucleic acid molecules are used inwhich the antibody coding sequences and any other desired sequences areflanked by regions that promote homologous recombination at a desiredsite in the genome, thus providing for intrachromosomal expression ofthe antibody encoding nucleic acids (Koller and Smithies, Proc. Natl.Acad. Sci. USA 86:8932-8935 (1989); Zijistra et al., Nature 342:435-438(1989). In specific embodiments, the expressed antibody molecule is asingle chain antibody; alternatively, the nucleic acid sequences includesequences encoding both the heavy and light chains, or fragmentsthereof, of the antibody.

Delivery of the nucleic acids into a patient may be either direct, inwhich case the patient is directly exposed to the nucleic acid ornucleic acid-carrying vectors, or indirect, in which case, cells arefirst transformed with the nucleic acids in vitro, then transplantedinto the patient. These two approaches are known, respectively, as invivo or ex vivo gene therapy.

In a specific embodiment, the nucleic acid sequences are directlyadministered in vivo, where it is expressed to produce the encodedproduct. This can be accomplished by any of numerous methods known inthe art, e.g., by constructing them as part of an appropriate nucleicacid expression vector and administering it so that they becomeintracellular, e.g., by infection using defective or attenuatedretrovirals or other viral vectors (see U.S. Pat. No. 4,980,286), or bydirect injection of naked DNA, or by use of microparticle bombardment(e.g., a gene gun; Biolistic, Dupont), or coating with lipids orcell-surface receptors or transfecting agents, encapsulating inliposomes, microparticles, or microcapsules, or by administering them inlinkage to a peptide which is known to enter the nucleus, byadministering it in linkage to a ligand subject to receptor-mediatedendocytosis (see, e.g., Wu and Wu, J. Biol. Chem., 262:4429-4432 (19870)(which can be used to target cell types specifically expressing thereceptors), etc. in another embodiment, nucleic acid-ligand complexescan be formed in which the ligand comprises a fusogenic viral peptide todisrupt endosomes, allowing the nucleic acid to avoid lysosomaldegradation. In yet another embodiment, the nucleic acid can be targetedin vivo for cell specific uptake and expression, by targeting a specificreceptor (see, e.g., PCT Publications WO 92/06180; WO 92/22635;WO92/20316; WO93/14188, WO 93/20221). Alternatively, the nucleic acidcan be introduced intracellularly and incorporated within host cell DNAfor expression, by homologous recombination (Koller and Smithies, Proc.Natl. Acad. Sci. USA 86:8932-8935 (1989); Zijistra et al., Nature342:435-438 (1989)).

In a specific embodiment, viral vectors that contain nucleic acidsequences encoding an antibody of the invention are used. For example, aretroviral vector can be used (see Miller et al., Meth. Enzymol.217:581-599 (1993)). These retroviral vectors contain the componentsnecessary for the correct packaging of the viral genome and integrationinto the host cell DNA. The nucleic acid sequences encoding the antibodyto be used in gene therapy are cloned into one or more vectors, whichfacilitates the delivery of the gene into a patient. More detail aboutretroviral vectors can be found in Boesen et al., Biotherapy 6:291-302(1994), which describes the use of a retroviral vector to deliver themdrl gene to hematopoietic stem cells in order to make the stem cellsmore resistant to chemotherapy. Other references illustrating the use ofretroviral vectors in gene therapy are: Clowes et al., J. Clin. Invest.93:644-651 (1994): Kiem et al., Blood 83:1467-1473 (1994); Salmons andGunzberg, Human Gene Therapy 4:129-141 (1993); and Grossman and Wilson,Curr. Opin. Gen. and Dev. 3:110-114 (1993).

Adenoviruses may also be used in the present invention. Adenoviruses areespecially attractive vehicles in the present invention for deliveringantibodies to respiratory epithelia. Adenoviruses naturally infectrespiratory epithelia. Other targets for adenovirus-based deliverysystems are liver, the central nervous system, endothelial cells, andmuscle. Adenoviruses have the advantage of being capable of infectingnon-dividing cells. Kozarsky and Wilson, Curr. Opin. Gen. Dev. 3:499-503(1993) present a review of adenovirus-based gene therapy. Boul et al.,Human Gene Therapy 5:3-10 (1994) demonstrated the use of adenovirusvectors to transfer genes to the respiratory epithelia of rhesusmonkeys. Other instances of the use of adenoviruses in gene therapy canbe found in Rosenfeld et al., Science 252:431-434 (1991); Rosenfield etal., Cell 68:143-155 (1992); Mastrangeli et al., J. Clin. Invest.91:335-234 (1993); PCT Publication WO94/12649; and Wang, et al., GeneTherapy 2:775-783 (1995). Adeno-associated virus (AAV) has also beenproposed for use in gene therapy (Walsh et al., Proc. Soc. Exp. Biol.Med. 204:289-300 (1993); U.S. Pat. Nos. 5,436,146; 6,632,670;6,642,051).

Another approach to gene therapy involves transferring a gene to cellsin tissue culture by such methods as electroporation, lipofection,calcium phosphate mediated transfection, or viral infection. Usually,the method of transfer includes the transfer of a selectable marker tothe cells. The cells are then placed under selection to isolate thosecells that have taken up and are expressing the transferred gene. Thosecells are then delivered to a patient.

In this embodiment, the nucleic acid is introduced into a cell prior toadministration in vivo of the resulting recombinant cell. Suchintroduction can be carried out by any method known in the art,including but not limited to transfection, electroporation,microinjection, infection with a viral or bacteriophage vectorcontaining the nucleic acid sequences, cell fusion, chromosome-mediatedgene transfer, microcell-mediated gene transfer, spheroplast fusion,etc. Numerous techniques are known in the art for the introduction offoreign genes into cells (see, e.g., Loeffier and Bahr, Meth. Enzymol,217:599-618 (1993); Cohen et al., Meth. Enzymol. 217:618-644 (1993);Cline, Pharmac. Ther. 29:69-92 m (1985) and may be used in accordancewith the present invention, provided that the necessary developmentaland physiological functions of the recipient cells are not disrupted.The technique should provide for the stable transfer of the nucleic acidto the cell, so that the nucleic acid is expressible by the cell andpreferably heritable and expressible by its cell progeny.

The resulting recombinant cells can be delivered to a patient by variousmethods known in the art. Recombinant blood cells (e.g., hematopoieticstem or progenitor cells) are preferably administered intravenously. Theamount of cells envisioned for use depends on the desired effect,patient state, etc., and can be determined by one skilled in the art.

Cells into which a nucleic acid can be introduced for purposes of genetherapy encompass any desired, available cell type, and include by arenot limited to epithelial cells, endothelial cells, kerathocytes,fibroblasts, muscle cells, hepatocytes; blood cells such as Tlymphocytes, B lymphocytes, monocytes, macrophages, neutrophils,eosinophils, megakaryocytes, granulocytes; various stem or progenitorcells, in particular hematopoietic stem or progenitor cells, e.g., asobtained from bone marrow, umbilical cord blood, peripheral blood, fetalliver, etc.

In a one embodiment, the cell used for gene therapy is autotogous to thepatient. Nucleic acid sequences encoding an antibody of the presentinvention are introduced into the cells such that they are expressibleby the cells or their progeny, and the recombinant cells are thenadministered in vivo for therapeutic effect. In a specific embodiment,stem or progenitor cells are used. Any stem and/or progenitor cellswhich can be isolated and maintained in vitro can potentially be used inaccordance with this embodiment of the present invention (see e.g., PCTPublication WO 94/08598; Stemple and Anderson, Cell 71:973-985 (1992);Rheinwald, Meth. Cell Bio. 21A:229 (1980); and Pittelkow and Scott, MayoClinic Proc. 61:771 (1986)).

Modulating Complement Pathway Component Expression by SIRNA

siRNAs have proven useful as a tool in studies of modulating geneexpression where traditional antagonists such as small molecules orantibodies may be less effective. (Shi Y., Trends in Genetics 19(10:9-12(2003)). In vitro synthesized, double stranded RNAs that are 21 to 23nucleotides in length can act as interfering RNAs (iRNAs) and canspecifically inhibit gene expression (Fire A., Trends in Genetics 391;806-810 (1999)). These iRNAs act by mediating degradation of theirtarget RNAs. Since they are under 30 nucleotides in length, they do nottrigger a cell antiviral defense mechanism. Such mechanisms includeinterferon production, and a general shutdown of host cell proteinsynthesis. Practically, siRNAs can be synthesized and then cloned intoDNA vectors. Such vectors can be transfected and made to express thesiRNA at high levels. The high level of siRNA expression is used to“knockdown” or significantly reduce the amount of protein produced in acell, and thus it is useful in experiments where overexpression of aprotein is believed to be linked to a disorder such as cancer. siRNAsare useful antagonists to complement pathway proteins by limitingcellular production of the antigen and inhibit activation of thecomplement cascade.

Peptidomimetics and Small Molecules

It is well-known to those normally skilled in the art that it ispossible to replace peptides with peptidomimetics. peptidomimetics aregenerally preferable as therapeutic agents to peptides owing to theirenhanced bioavailability and relative lack of attack from proteolyticenzymes. Techniques of molecular modeling may be used to design apeptidomimetics which mimic the structure of the complement relatedpeptides disclosed herein. Accordingly, the present invention alsoprovides peptidomimetics and other lead compounds which can beidentified based on the data obtained from structural analysis of thecomplement pathway protein. A potential Factor D analog may be examinedthrough the use of computer modeling using a docking program such asGRAM, DOCK, or AUTODOCK. This procedure can include computer fitting ofpotential Factor D analogs. Computer programs can also be employed toestimate the attraction, repulsion, and steric hindrance of an analog toa potential binding site. Generally the tighter the fit (e.g., the lowerthe steric hindrance, and/or the greater the attractive force) the morepotent the potential drug will be since these properties are consistentwith a tighter binding constant. Furthermore, the more specificity inthe design of a potential drug the more likely that the drug will notinterfere with other properties of the expression system. The willminimize potential side-effects due to unwanted interactions with otherproteins.

Initially a potential Factor D analog could be obtained by screening arandom peptide library produced by a recombinant bacteriophage, forexample, or a chemical library. An analog ligand selected in this mannercould be then be systematically modified by computer modeling programsuntil one or more promising potential ligands are identified.

Such computer modeling allows the selection of a finite number ofrational chemical modifications, as opposed to the countless number ofessentially random chemical modifications that could be made, and ofwhich any one might lead to a useful drug. Thus through the use of thethree-dimensional structure disclosed herein and computer modeling, alarge number of compounds is rapidly screened and a few likelycandidates can be determined without the laborious synthesis of untoldnumbers of compounds.

Once a potential Factor D analog is identified it can be either selectedfrom a library of chemicals commercially available from most largechemical companies including Merck, GlaxoWelcome, Bristol Meyers Squib,Monsanto/Searle, Eli Lilly, Novartis and Pharmacia UpJohn, oralternatively the potential ligand is synthesized de novo. As mentionedabove, the de novo synthesis of one or even a relatively small group ofspecific compounds is reasonable in the art of drug design.

Alternatively, based on the molecular structures of the variable regionsof the anti-Factor D antibodies, one could use molecular modeling andrational molecular design to generate and screen small molecules whichmimic the molecular structures of the binding region of the antibodiesand inhibit the activities of Factor D. These small molecules can bepeptides, peptidomimetics, oligonucleotides, or organic compounds.

Example Efficacy of Antibody in a Laser-Induced Choroidal NeoyAscularization (CNV) as a Model of Wet AMD

The efficacy of intraocular injections of an antibody may be tested in alaser-injury CNV model as described earlier by Krzystolik M G et al.(Arch Ophthalm. 2002; 120: 338-346). This model may be used to test theefficacy of any drug candidate for the prevention and/or amelioration ofAMD. This laser induced CNV model uses argon green laser to induce CNVin the monkey macula. There is a good correlation between the number ofCNV lesions with significant angiographic leakage.

There are two phases of the studies: Phase 1, the prevention phase,involves the initiation of antibody treatment before laser induction ofthe CNV and 1 week after exposure to the laser to inhibit the formationof CNV, which typically appears 2 to 3 weeks after laser injury. Phase2, the treatment phase, is initiated on day 42 (3 weeks after laserinjury) when CNV lesions would be expected in the control eyes fromphase 1. Phase 2 assesses the effect of treatment on attenuating theextent and leakiness of existing CNV lesions.

Ten cynomolgus monkeys (Macaca fasicularis) are typically used in astudy of this type. The monkeys are anesthetized for all procedures withintramuscular injections of, e.g., ketamine hydrochloride (20 mg/kg);acepromazine maleate (0.125 mg/kg); and atropine sulfate (0.125 mg/kg).Supplemental anesthesia of 5 to 6 mg/kg of ketamine hydrochloride may beadministered as needed. In addition, 0.5% proparacaine hydrochloride istypically used for topical anesthesia. Supplemental anesthesia, withintravenous peutobarbital sodium solution (5 mg/kg), may be administeredbefore enucleation. Animals are euthanized following theexperimentation.

Antibody Treatment

The antibody to be tested is administered in a physiological buffer at aconcentration of about e.g., 10 μg/μL. The control eye is injected witha vehicle consisting of all components except the antibody to be tested.Intraocular injections of about, e.g., 50 μL per eye with eitherantibody or vehicle is performed on each eye, respectively, through thepars plana using a 30-gauge needle and tuberculin syringe afterinstilling topical anesthesia and 5% providone iodine solution. Theantibody is withdrawn from a vial through a 5-μm filter, and a new(sharp) 30-gauge needle is used for the intraocular injection. After theinjection, a bacteriocidal ophthalmic ointment such as bacitracin isinstilled in the fornices. The injection sites are typically varied toavoid trauma to the sclera.

In phase 1, the right or left eye of each animal is randomly assigned toreceive intraocular injections of antibody at a dose of about, e.g., 500μg (50 μL per eye), and this eye is termed the prevention eye. The doseused may be determined based on a safety and toxicology study prior tothis efficacy study, or by other clinically appropriate means. The othereye is assigned to receive intraocular injections of vehicle and istermed the control eye. Both eyes of each animal typically receive twointraocular injections with either the antibody to be tested or thevehicle alone on days 0 and 14 before laser treatment. On day 21, alleyes undergo an argon green laser photocoagulation to induce CNVlesions. On day 28, one week after laser induction, the prevention eyereceives another injection of antibody and the control eye receivedvehicle. Phase 2 of the study begins on day 42 or 3 weeks after laserinduction, when CNV is expected to have developed. Following fluoresceinangiography on day 42, both eyes of each animal will receive intraocularinjections of antibody at a dose of about, e.g., 500 μg (50 μL per eye),and this is repeated on day 56.

Induction of Experimental CNV

The CNV membranes are induced in the macula of cynomolgus monkeys withargon green laser burns (Coherent Argon Dye Laser 920; Coherent MedicalLaser, Palo Alto, Calif.) using a slit-lamp and a plano fundus contactlens. Nine lesions are symmetrically placed in the macula of each eye bya masked surgeon. The laser variables include a 50- to 100-μm spot size,0.1-second duration, and power ranging from 350 to 700 mW. The powerused is determined by the laser's ability to produce a blister and asmall hemorrhage under the power chosen. If no hemorrhage is noted, anadditional laser spot will be placed adjacent to the first spotfollowing the same laser procedure. Color photographs and fluoresceinangiography are typically used to detect and measure the extent andleakiness of the CNV. However, any method capable of measuringlaser-induced CNV and its associated effects may be used.

Ocular Examinations

The eyes of the animals are checked for relative pupillary afferentdefect and then dilated with 2.5% phenylephrine hydorchloride and 0.8%tropicamide. Both eyes are examined using slitlamp biomicroscopy andindirect ophthalmoscopy on days 0, 14, 28, 42, and 56 (before antibodyinjection); days 1, 15, 29, 43, and 57 (after injection); day 21 (beforelaser); days 35 and 49 (intermediate days); and day 63 (enucleation anddeath).

Color Photography and Fluorescein Angiography

Fundus photography is typically performed on all animals on the samedays as the ocular examination. Photographs may be obtained with afundus camera (Canon Fundus CF-60Z; Canon USA Inc, Lake Success, N.Y.)and 35-mm film, but any photography device may be used.

The Imagenet Digital Angiography System (Topcon 501 A and Imagenetsystem; Topcon America Corp, Paramus, N.J.) may be used for fluoresceinangiography. Red-free photographs of both eyes is typically obtainedfollowed by fluorescein angiography using 0.1 mL/kg of body weight of10% sodium fluorescein (Akorn Inc. Abita Springs, La.) at a rate of 1mL/s. Following the fluorescein injection, a rapid series of images isobtained in the first minute of the posterior pole of first the righteye and then the left eye. Additional pairs of images are typicallyobtained at approximately 1 to 2 and 5 minutes. Between 2 and 5 minutes,two images of the midperipheral fields (temporal and nasal) are taken ofeach eye. Fluorescein angiography is performed at baseline (day 0) anddays 7, 14, 29, 42, 49, 57, and 63.

Analysis of Ophthalmic Data

Photographs and angiograms are evaluated for evidence of angiographicleakage, hemorrhages, or any other abnormalities. The fundus hemorrhagesare graded based on a grading system with retinal hemorrhages thatinvolves less than 3 disc areas defined as grade 1, hemorrhages between3 and 6 disc areas defined as grade 2, and hemorrhages of more than 6disc areas defined as grade 3. The association of hemorrhages with CNVmembranes or the laser induction site is also assessed. Clinicallysignificant bleeding is defined as any fundus hemorrhage greater than orequal to a 6-disc area.

Ocular inflammation is also assessed using a slit-lamp biomicroscopy.Anterior chamber and vitreal cells are counted with a 2-mm slit-lamp ata high magnification and graded using the schema of the American Academyof Ophthalmology. The CNV lesions are graded by reviewing fluoresceinangiograms performed on days 35, 42, 49, 56, and 63 by experiencedexaminers, typically two, who grade by consensus opinion. The CNVlesions are graded according to the following scheme, using standardizedangiographs for comparison. Grade 1 lesions have no hyperfluorescence.Grade 2 lesions exhibit hyperfluorescence without leakage. Grade 3lesions show hyperfluorescence in the early or mid-transit images andlate leakage. Grade 4 lesions show bright hyperfluorescence in thetransit and late leakage beyond the treated areas. Grade 4 lesions aredefined as clinically significant.

Statistical analysis may be performed using the Population-AggregatedPanel Data with Generalized Estimating Equations nd the incidence rateratio (IRR). The incidence rate is usually defined as the number ofgrade 4 lesions that occur during a given interval divided by the totalnumber of lesions induced. In phase 1, the IRR refers to the ratio ofincidence rate of grade 4 lesions in the prevention eyes to theincidence rate in control eyes. An IRR of 1 signifies no differencebetween incidence rates. A number much smaller than 1 will indicate areduction in the incidence of grade 4 lesions in the prevention groupvs. control group. In phase 2, the incidence of grade 4 lesions in thecontrol eyes vs. the treatment eyes is compared. This means that theincidence of grade 4 lesions is compared over time in the set of eyesthat are first assigned to the control group but on days 42 and 56 aretreated with antibody and become treatment eyes.

Screen for Agents Useful in the Treatment of AMD

The study and treatment of age-related macular degeneration (AMD) can beaccomplished using a new animal model comprising mice deficient eitherin monocyte chemoattractant protein-1 (Ccl-2; also known as MCP-1) orits cognate C—C chemokine receptor-2 (Ccr-2) (Ambati, J. et al. Nat Med.2003 November; 9(11):1390-7. Epob 2003 Oct. 19). These mice developcardinal features of AMD, including accumulation of lipofuscin in anddrusen beneath the retinal pigmented epithelium (RPE), photoreceptoratrophy and choroidal neovascularization (CNV).

Treatment of these mice with a desired agent may allow assessment of theefficacy of such an agent for its efficacy in treating AMD.

1.-27. (canceled)
 28. A method for preventing or ameliorating an oculardisease that involves complement activation, comprising administering aneffective amount of a complement inhibitor to a subject in need thereof.29. The method of claim 28, wherein the ocular disease is selected fromthe group consisting of macular degeneration, diabetic retinopathy, andocular angiogenesis.
 30. The method of claim 28, wherein the subjectrequires inhibition of ocular neovascularization that affects thechoroid, retinal pigmented epithelium, or retinal tissue.
 31. The methodof claim 28, wherein the complement inhibitor inhibits the alternativecomplement pathway.
 32. The method of claim 28, wherein the complementinhibitor is Factor H, or a functional peptide thereof or apeptidomimetic thereof.
 33. The method according to claim 28, whereinthe complement inhibitor is an antibody or an antigen-binding fragmentthereof.
 34. The method according to claim 33 wherein the antibody orthe antigen-binding fragment thereof specifically binds to Factor D,properdin, Factor B, Factor Ba, Factor Bb, C2, C2a, C3a, C5, C5a, C5b,C6, C7, C8, C9 or C5b-9.
 35. The method according to claim 33, whereinthe antibody is monoclonal antibody 166-32 produced from the hybridomadeposited with the ATCC and designated HB
 12476. 36. The methodaccording to claim 33, wherein the antibody or the antigen-bindingfragment thereof specifically binds to the same epitope as monoclonalantibody 166-32 produced from the hybridoma deposited with the ATCC anddesignated HB
 12476. 37. The method according to claim 33, wherein theantibody is a humanized monoclonal antibody derived from monoclonalantibody 166-32 produced from the hybridoma deposited with the ATCC anddesignated HB
 12476. 38. The method according to claim 33, wherein theantibody or the antigen-binding fragment thereof specifically binds tocomplement component C5a.
 39. The method according to claim 38, whereinthe antibody is 137-26 produced from the hybridoma deposited with theATCC and designated PTA-3650.
 40. The method according to claim 38,wherein the antibody or the antigen-binding fragment thereof binds tothe same epitope as monoclonal antibody 137-26 produced from thehybridoma deposited with the ATCC and designated PTA-3650.
 41. Themethod of claim 28, wherein the complement inhibitor is administered byintraocular administration, intravitreal administration, orsubconjunctival administration, parenteral administration, intradermaladministration, intramuscular administration, intraperitonealadministration, intravenous administration, subantaneous administration,intranasal administration, oral administration, enteral administration,topical administration, intrathecal administration, intraventricularadministration, epidural, inhalation, a biocompatible or bioerodablesustained release implant, or implantation of an infusion pump.
 42. Themethod according to claim 28, wherein the complement inhibitor isadministered in an eye wash solution, an eye ointment, an eye shield oran eye drop solution.
 43. The method according to claim 28, furthercomprising the step of administering an immunomodulatory compound, animmunosuppressive compound, an anti-inflammatory compound, or ananti-angiogenic compound, to said subject.
 44. The method of claim 28,further comprising administering to the subject an anti-angiogenesistherapy targeting vascular endothelial growth factor (VEGF).
 45. Themethod of claim 28, further comprising administering to the subject asteroid.
 46. A method of preventing or ameliorating an ocular diseasethat involves complement activation, comprising administering an siRNAspecific for a complement pathway protein to a subject in need thereof.47. A method of preventing or ameliorating an ocular disease thatinvolves complement activation, comprising administering a nucleic acidencoding a complement inhibitor to a subject in need thereof.
 48. Themethod of claim 29, wherein the macular degeneration is age-relatedmacular degeneration.
 49. The method claim 48, wherein the age-relatedmacular degeneration is the dry form of age-related maculardegeneration.